<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2025 Workiva-->
<!--r:268d30be-eab7-4b75-a019-8d24580e7f04,g:2ec1a744-520c-4b1f-a98c-c846b2b61c36-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://www.nautilus.bio/role/CondensedConsolidatedStatementsofStockholdersEquity" xlink:type="simple" xlink:href="naut-20250930.xsd#CondensedConsolidatedStatementsofStockholdersEquity"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/CondensedConsolidatedStatementsofStockholdersEquity" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_9a775b13-ffc7-426c-964e-af5ef66e2d76" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_5bdedf45-3e3b-4f3e-943e-b9ff49d91e2e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_9a775b13-ffc7-426c-964e-af5ef66e2d76" xlink:to="loc_us-gaap_StatementTable_5bdedf45-3e3b-4f3e-943e-b9ff49d91e2e" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_7fc38484-192a-4d82-af90-9fd2fda3db6e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_5bdedf45-3e3b-4f3e-943e-b9ff49d91e2e" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_7fc38484-192a-4d82-af90-9fd2fda3db6e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_7fc38484-192a-4d82-af90-9fd2fda3db6e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_7fc38484-192a-4d82-af90-9fd2fda3db6e" xlink:to="loc_us-gaap_EquityComponentDomain_7fc38484-192a-4d82-af90-9fd2fda3db6e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_95c641f8-aed7-4fc3-bc52-63e7ef292156" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_7fc38484-192a-4d82-af90-9fd2fda3db6e" xlink:to="loc_us-gaap_EquityComponentDomain_95c641f8-aed7-4fc3-bc52-63e7ef292156" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_20dc5096-8ebc-4d61-934d-31fa6e466c91" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_95c641f8-aed7-4fc3-bc52-63e7ef292156" xlink:to="loc_us-gaap_CommonStockMember_20dc5096-8ebc-4d61-934d-31fa6e466c91" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalMember_fc17a121-a641-4ca6-85f5-9d933c5bb33b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_95c641f8-aed7-4fc3-bc52-63e7ef292156" xlink:to="loc_us-gaap_AdditionalPaidInCapitalMember_fc17a121-a641-4ca6-85f5-9d933c5bb33b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_a458a71d-fb61-4fd9-a2d5-d872e2fc7a04" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_95c641f8-aed7-4fc3-bc52-63e7ef292156" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_a458a71d-fb61-4fd9-a2d5-d872e2fc7a04" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_2432f078-9ab5-46b0-9698-950ef66baf6d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_95c641f8-aed7-4fc3-bc52-63e7ef292156" xlink:to="loc_us-gaap_RetainedEarningsMember_2432f078-9ab5-46b0-9698-950ef66baf6d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a775b13-ffc7-426c-964e-af5ef66e2d76" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_e29619e5-f4fb-4788-8beb-ed77f9a8e5e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_e29619e5-f4fb-4788-8beb-ed77f9a8e5e7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_9ac22675-003d-4f71-a688-82b594022740" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_StockholdersEquity_9ac22675-003d-4f71-a688-82b594022740" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_92e980ed-f8b4-481a-a492-41e1fe503325" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_92e980ed-f8b4-481a-a492-41e1fe503325" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_b43e3cdf-6477-4c3f-b6e4-e0e9221feab0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_b43e3cdf-6477-4c3f-b6e4-e0e9221feab0" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_fc486a4b-bd37-4ac4-814e-7d6087bcf171" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_fc486a4b-bd37-4ac4-814e-7d6087bcf171" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan_7d5cca76-a7c7-4b9c-b75f-0b23ca3700a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan_7d5cca76-a7c7-4b9c-b75f-0b23ca3700a5" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_b7a4182b-c40b-450f-9635-b51923d6a57b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_b7a4182b-c40b-450f-9635-b51923d6a57b" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_53c19d42-657d-44fd-88dd-6094c0805a0a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_53c19d42-657d-44fd-88dd-6094c0805a0a" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_9cc7bc68-020a-4cd2-800b-9503e9dc3ea4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_66b210b3-e174-4e0d-8512-819063522c9d" xlink:to="loc_us-gaap_NetIncomeLoss_9cc7bc68-020a-4cd2-800b-9503e9dc3ea4" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_a4af6941-89c5-46d8-904b-54c6ee3f33c5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_08b854c2-4f93-42d7-8e44-99713a9a71e4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/DescriptionofBusinessandBasisofPresentationDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#DescriptionofBusinessandBasisofPresentationDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/DescriptionofBusinessandBasisofPresentationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_d52ad157-b915-4e32-9243-523630c02515" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_d52ad157-b915-4e32-9243-523630c02515" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_ee8f64d3-0b5b-4ae0-9b77-327a5d8e4c6c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_d52ad157-b915-4e32-9243-523630c02515" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_ee8f64d3-0b5b-4ae0-9b77-327a5d8e4c6c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_ee8f64d3-0b5b-4ae0-9b77-327a5d8e4c6c_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_ee8f64d3-0b5b-4ae0-9b77-327a5d8e4c6c" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_ee8f64d3-0b5b-4ae0-9b77-327a5d8e4c6c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_598adc6c-18cc-485b-9394-bc3747b8360e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_ee8f64d3-0b5b-4ae0-9b77-327a5d8e4c6c" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_598adc6c-18cc-485b-9394-bc3747b8360e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PrivatePlacementMember_fba4fb5e-e82a-4b61-9d79-1e410e425dab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PrivatePlacementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_598adc6c-18cc-485b-9394-bc3747b8360e" xlink:to="loc_us-gaap_PrivatePlacementMember_fba4fb5e-e82a-4b61-9d79-1e410e425dab" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_9c87b185-52c4-4fce-95b4-0e72d2195ca8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_9c87b185-52c4-4fce-95b4-0e72d2195ca8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockPricePerShare_40ff56f5-22d2-4ceb-83de-5bd11f29c00c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockPricePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_us-gaap_SaleOfStockPricePerShare_40ff56f5-22d2-4ceb-83de-5bd11f29c00c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_3c244cd4-1950-4c79-a20b-1c5b8ac48b29" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_3c244cd4-1950-4c79-a20b-1c5b8ac48b29" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsAccumulatedDeficit_60efa071-64fd-4023-9b48-b56b1409beea" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsAccumulatedDeficit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_us-gaap_RetainedEarningsAccumulatedDeficit_60efa071-64fd-4023-9b48-b56b1409beea" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_ProceedsFromSaleOfStockAndReverseRecapitalizationTransaction_e7719154-6a78-4af5-9e61-0a99e165b787" xlink:href="naut-20250930.xsd#naut_ProceedsFromSaleOfStockAndReverseRecapitalizationTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_naut_ProceedsFromSaleOfStockAndReverseRecapitalizationTransaction_e7719154-6a78-4af5-9e61-0a99e165b787" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_PaymentsOfReverseRecapitalizationTransactionCosts_3e669fd5-ea31-4d07-9707-3c0a337a8d9f" xlink:href="naut-20250930.xsd#naut_PaymentsOfReverseRecapitalizationTransactionCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_naut_PaymentsOfReverseRecapitalizationTransactionCosts_3e669fd5-ea31-4d07-9707-3c0a337a8d9f" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashCashEquivalentsAndShortTermInvestments_2eee982d-cdeb-4928-add8-4b601ecfae46" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashCashEquivalentsAndShortTermInvestments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_1e19a5b4-967e-49a0-8dad-356fa8ad8032" xlink:to="loc_us-gaap_CashCashEquivalentsAndShortTermInvestments_2eee982d-cdeb-4928-add8-4b601ecfae46" xlink:type="arc" order="6"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/FairValueMeasurementsScheduleofAssetsCarriedatFairValueandMeasuredonaRecurringBasisDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#FairValueMeasurementsScheduleofAssetsCarriedatFairValueandMeasuredonaRecurringBasisDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/FairValueMeasurementsScheduleofAssetsCarriedatFairValueandMeasuredonaRecurringBasisDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_d5d5c64f-90af-430f-bf9b-4403e081536d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_be70e1b0-2f00-4746-bfbd-738ef6447dbe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_d5d5c64f-90af-430f-bf9b-4403e081536d" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_be70e1b0-2f00-4746-bfbd-738ef6447dbe" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_b8e7bea6-4f3a-4d4c-8931-d498bfc458a0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_be70e1b0-2f00-4746-bfbd-738ef6447dbe" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_b8e7bea6-4f3a-4d4c-8931-d498bfc458a0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b8e7bea6-4f3a-4d4c-8931-d498bfc458a0_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_b8e7bea6-4f3a-4d4c-8931-d498bfc458a0" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b8e7bea6-4f3a-4d4c-8931-d498bfc458a0_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_e8b9ae9d-36a3-4a0e-b302-6a7d51b2a4cc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_b8e7bea6-4f3a-4d4c-8931-d498bfc458a0" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_e8b9ae9d-36a3-4a0e-b302-6a7d51b2a4cc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_a01fd5a4-33e3-4b48-be61-825b63996a50" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_e8b9ae9d-36a3-4a0e-b302-6a7d51b2a4cc" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_a01fd5a4-33e3-4b48-be61-825b63996a50" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_304d4aa4-45c5-4d6a-8fe0-fa85830e1bfe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_e8b9ae9d-36a3-4a0e-b302-6a7d51b2a4cc" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_304d4aa4-45c5-4d6a-8fe0-fa85830e1bfe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementFrequencyAxis_7f50061d-bd36-4e1a-a9dc-930a6c9a65cd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByMeasurementFrequencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_be70e1b0-2f00-4746-bfbd-738ef6447dbe" xlink:to="loc_us-gaap_FairValueByMeasurementFrequencyAxis_7f50061d-bd36-4e1a-a9dc-930a6c9a65cd" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementFrequencyDomain_7f50061d-bd36-4e1a-a9dc-930a6c9a65cd_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementFrequencyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementFrequencyAxis_7f50061d-bd36-4e1a-a9dc-930a6c9a65cd" xlink:to="loc_us-gaap_FairValueMeasurementFrequencyDomain_7f50061d-bd36-4e1a-a9dc-930a6c9a65cd_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementFrequencyDomain_24bb174a-0458-4124-8b87-34ce9b5108cc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementFrequencyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementFrequencyAxis_7f50061d-bd36-4e1a-a9dc-930a6c9a65cd" xlink:to="loc_us-gaap_FairValueMeasurementFrequencyDomain_24bb174a-0458-4124-8b87-34ce9b5108cc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsRecurringMember_21518c10-4a3a-4d1e-967f-77c14949154d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsRecurringMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementFrequencyDomain_24bb174a-0458-4124-8b87-34ce9b5108cc" xlink:to="loc_us-gaap_FairValueMeasurementsRecurringMember_21518c10-4a3a-4d1e-967f-77c14949154d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_f4e52a16-462d-4b4c-b117-2fca2074f0d8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_be70e1b0-2f00-4746-bfbd-738ef6447dbe" xlink:to="loc_us-gaap_FinancialInstrumentAxis_f4e52a16-462d-4b4c-b117-2fca2074f0d8" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f4e52a16-462d-4b4c-b117-2fca2074f0d8_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_f4e52a16-462d-4b4c-b117-2fca2074f0d8" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f4e52a16-462d-4b4c-b117-2fca2074f0d8_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_c137b348-185a-4281-a724-cb4fd6509616" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_f4e52a16-462d-4b4c-b117-2fca2074f0d8" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_c137b348-185a-4281-a724-cb4fd6509616" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MutualFundMember_f2ac8c10-c924-4264-bc86-5f0408981a94" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MutualFundMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_c137b348-185a-4281-a724-cb4fd6509616" xlink:to="loc_us-gaap_MutualFundMember_f2ac8c10-c924-4264-bc86-5f0408981a94" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasurySecuritiesMember_a979743b-2c05-4620-962d-71fb9a90c6a2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasurySecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_c137b348-185a-4281-a724-cb4fd6509616" xlink:to="loc_us-gaap_USTreasurySecuritiesMember_a979743b-2c05-4620-962d-71fb9a90c6a2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_a74548f2-046b-42b4-b1ed-8d225fb4ceff" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_c137b348-185a-4281-a724-cb4fd6509616" xlink:to="loc_us-gaap_CommercialPaperMember_a74548f2-046b-42b4-b1ed-8d225fb4ceff" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USGovernmentAgenciesDebtSecuritiesMember_62ef5f4d-217e-46ec-af74-7644a00ab8ba" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_c137b348-185a-4281-a724-cb4fd6509616" xlink:to="loc_us-gaap_USGovernmentAgenciesDebtSecuritiesMember_62ef5f4d-217e-46ec-af74-7644a00ab8ba" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationAxis_d9298908-6a6a-4140-a215-38d85e15e871" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_be70e1b0-2f00-4746-bfbd-738ef6447dbe" xlink:to="loc_us-gaap_BalanceSheetLocationAxis_d9298908-6a6a-4140-a215-38d85e15e871" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_d9298908-6a6a-4140-a215-38d85e15e871_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_d9298908-6a6a-4140-a215-38d85e15e871" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_d9298908-6a6a-4140-a215-38d85e15e871_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_16f53072-1f46-4070-b1e6-215c8885d1c8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_d9298908-6a6a-4140-a215-38d85e15e871" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_16f53072-1f46-4070-b1e6-215c8885d1c8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsMember_14f93294-0afa-4ce6-99c3-515a43745f95" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_16f53072-1f46-4070-b1e6-215c8885d1c8" xlink:to="loc_us-gaap_CashAndCashEquivalentsMember_14f93294-0afa-4ce6-99c3-515a43745f95" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShortTermInvestmentsMember_abe3b5c8-58e7-4bbc-93f6-5487de19b88c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShortTermInvestmentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_16f53072-1f46-4070-b1e6-215c8885d1c8" xlink:to="loc_us-gaap_ShortTermInvestmentsMember_abe3b5c8-58e7-4bbc-93f6-5487de19b88c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_LongTermInvestmentsMember_52763976-eb14-4513-af92-71209d902688" xlink:href="naut-20250930.xsd#naut_LongTermInvestmentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_16f53072-1f46-4070-b1e6-215c8885d1c8" xlink:to="loc_naut_LongTermInvestmentsMember_52763976-eb14-4513-af92-71209d902688" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_1eee3c96-dffb-4d87-9742-713435a9d7d4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_d5d5c64f-90af-430f-bf9b-4403e081536d" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_1eee3c96-dffb-4d87-9742-713435a9d7d4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax_ec0daf3f-0aed-4a92-bd92-1a86d977001b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_d5d5c64f-90af-430f-bf9b-4403e081536d" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax_ec0daf3f-0aed-4a92-bd92-1a86d977001b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax_58d250aa-f097-4aed-801e-d55b410b78b3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_d5d5c64f-90af-430f-bf9b-4403e081536d" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax_58d250aa-f097-4aed-801e-d55b410b78b3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_43cea32a-938f-4691-8d9b-6d933c2b5353" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_d5d5c64f-90af-430f-bf9b-4403e081536d" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_43cea32a-938f-4691-8d9b-6d933c2b5353" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/FairValueMeasurementsUnrealizedLossesandFairValuesofAvailableforSaleSecuritiesinanUnrealizedLossPositionDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#FairValueMeasurementsUnrealizedLossesandFairValuesofAvailableforSaleSecuritiesinanUnrealizedLossPositionDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/FairValueMeasurementsUnrealizedLossesandFairValuesofAvailableforSaleSecuritiesinanUnrealizedLossPositionDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_804707d0-161d-452f-b720-402e1381035b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_84cec53b-6896-4b17-8a1a-a433ebd049f6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_804707d0-161d-452f-b720-402e1381035b" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_84cec53b-6896-4b17-8a1a-a433ebd049f6" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_21c245dc-bef2-41d7-ab46-bd27d6099d30" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_84cec53b-6896-4b17-8a1a-a433ebd049f6" xlink:to="loc_us-gaap_FinancialInstrumentAxis_21c245dc-bef2-41d7-ab46-bd27d6099d30" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_21c245dc-bef2-41d7-ab46-bd27d6099d30_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_21c245dc-bef2-41d7-ab46-bd27d6099d30" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_21c245dc-bef2-41d7-ab46-bd27d6099d30_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_0cf606d4-8352-42e3-b69a-dce37369ca3c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_21c245dc-bef2-41d7-ab46-bd27d6099d30" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_0cf606d4-8352-42e3-b69a-dce37369ca3c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasurySecuritiesMember_7d5c0140-9cbc-4fbb-a9be-2c24f3003977" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasurySecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_0cf606d4-8352-42e3-b69a-dce37369ca3c" xlink:to="loc_us-gaap_USTreasurySecuritiesMember_7d5c0140-9cbc-4fbb-a9be-2c24f3003977" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_8767c44d-3915-453a-a393-a5c9a9b4abf7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_0cf606d4-8352-42e3-b69a-dce37369ca3c" xlink:to="loc_us-gaap_CommercialPaperMember_8767c44d-3915-453a-a393-a5c9a9b4abf7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USGovernmentAgenciesDebtSecuritiesMember_239c102c-5927-4dd6-b2ad-419f203ba91d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_0cf606d4-8352-42e3-b69a-dce37369ca3c" xlink:to="loc_us-gaap_USGovernmentAgenciesDebtSecuritiesMember_239c102c-5927-4dd6-b2ad-419f203ba91d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionAccumulatedLossAbstract_f85d3d0f-0678-4138-afa3-f196f65f6451" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionAccumulatedLossAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_804707d0-161d-452f-b720-402e1381035b" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionAccumulatedLossAbstract_f85d3d0f-0678-4138-afa3-f196f65f6451" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsAccumulatedLoss_d8a59bfd-8def-404c-b654-0b3f04e4a476" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsAccumulatedLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionAccumulatedLossAbstract_f85d3d0f-0678-4138-afa3-f196f65f6451" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsAccumulatedLoss_d8a59bfd-8def-404c-b654-0b3f04e4a476" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLongerAccumulatedLoss_bfa851bc-a4d9-41d1-8ee7-427e2abc61b1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLongerAccumulatedLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionAccumulatedLossAbstract_f85d3d0f-0678-4138-afa3-f196f65f6451" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLongerAccumulatedLoss_bfa851bc-a4d9-41d1-8ee7-427e2abc61b1" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss_9ff32b6b-62f3-4f34-9af9-cb47a4efd190" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionAccumulatedLossAbstract_f85d3d0f-0678-4138-afa3-f196f65f6451" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss_9ff32b6b-62f3-4f34-9af9-cb47a4efd190" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueAbstract_4e457054-9f55-4d55-8d38-0160ec45f078" xlink:href="naut-20250930.xsd#naut_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_804707d0-161d-452f-b720-402e1381035b" xlink:to="loc_naut_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueAbstract_4e457054-9f55-4d55-8d38-0160ec45f078" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months_ea875c16-407e-4441-8cb5-5e4a77fffa63" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_naut_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueAbstract_4e457054-9f55-4d55-8d38-0160ec45f078" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months_ea875c16-407e-4441-8cb5-5e4a77fffa63" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger_8f112865-f217-4233-8761-6f3503e94140" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_naut_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueAbstract_4e457054-9f55-4d55-8d38-0160ec45f078" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger_8f112865-f217-4233-8761-6f3503e94140" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition_9d6cab0f-2d01-4aeb-a2f2-953be8fee7bb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_naut_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueAbstract_4e457054-9f55-4d55-8d38-0160ec45f078" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition_9d6cab0f-2d01-4aeb-a2f2-953be8fee7bb" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/CompositionofCertainCondensedConsolidatedFinancialStatementLineItemsScheduleofPropertyandEquipmentNetDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#CompositionofCertainCondensedConsolidatedFinancialStatementLineItemsScheduleofPropertyandEquipmentNetDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/CompositionofCertainCondensedConsolidatedFinancialStatementLineItemsScheduleofPropertyandEquipmentNetDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentLineItems_26fac7f4-e2c1-4f64-8f46-0ca0574ef62a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_44f3bbdd-ed0e-4cf7-b119-cbbde3f4f45c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_26fac7f4-e2c1-4f64-8f46-0ca0574ef62a" xlink:to="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_44f3bbdd-ed0e-4cf7-b119-cbbde3f4f45c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_495b15a2-566b-4bda-b161-959578a47678" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_44f3bbdd-ed0e-4cf7-b119-cbbde3f4f45c" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_495b15a2-566b-4bda-b161-959578a47678" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_495b15a2-566b-4bda-b161-959578a47678_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_495b15a2-566b-4bda-b161-959578a47678" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_495b15a2-566b-4bda-b161-959578a47678_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_495b15a2-566b-4bda-b161-959578a47678" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_LaboratoryEquipmentMember_c2592689-bf45-4e1a-ac0c-6e5ce7cff1c1" xlink:href="naut-20250930.xsd#naut_LaboratoryEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:to="loc_naut_LaboratoryEquipmentMember_c2592689-bf45-4e1a-ac0c-6e5ce7cff1c1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LeaseholdImprovementsMember_545f7a62-0aaa-466c-8443-392cf5a962a7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LeaseholdImprovementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:to="loc_us-gaap_LeaseholdImprovementsMember_545f7a62-0aaa-466c-8443-392cf5a962a7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComputerEquipmentMember_d790995c-252a-4a82-bb80-52729bfa2bbd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComputerEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:to="loc_us-gaap_ComputerEquipmentMember_d790995c-252a-4a82-bb80-52729bfa2bbd" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_FurnitureFixturesAndOfficeEquipmentMember_798bcbb7-ff62-4764-8835-d7206f783abf" xlink:href="naut-20250930.xsd#naut_FurnitureFixturesAndOfficeEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:to="loc_naut_FurnitureFixturesAndOfficeEquipmentMember_798bcbb7-ff62-4764-8835-d7206f783abf" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_PrototypeEquipmentMember_2a0e587b-b27b-4e88-b8f5-0b31153a0611" xlink:href="naut-20250930.xsd#naut_PrototypeEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:to="loc_naut_PrototypeEquipmentMember_2a0e587b-b27b-4e88-b8f5-0b31153a0611" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConstructionInProgressMember_c110780e-c8d0-496e-bf02-203977d047cd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConstructionInProgressMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_2d51cfee-5967-49e2-92cf-8c12d983f7c3" xlink:to="loc_us-gaap_ConstructionInProgressMember_c110780e-c8d0-496e-bf02-203977d047cd" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentGross_bf0cf029-e587-41f5-818b-0e19091eaa21" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_26fac7f4-e2c1-4f64-8f46-0ca0574ef62a" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentGross_bf0cf029-e587-41f5-818b-0e19091eaa21" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_2d4de230-14ea-4031-ae00-9a230e5055c2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_26fac7f4-e2c1-4f64-8f46-0ca0574ef62a" xlink:to="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_2d4de230-14ea-4031-ae00-9a230e5055c2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentNet_8804786b-51b8-42af-b847-868a055ba4e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_26fac7f4-e2c1-4f64-8f46-0ca0574ef62a" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentNet_8804786b-51b8-42af-b847-868a055ba4e7" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/CommonStockScheduleofCommonStockReservedforFutureIssuanceDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#CommonStockScheduleofCommonStockReservedforFutureIssuanceDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/CommonStockScheduleofCommonStockReservedforFutureIssuanceDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockLineItems_658fbef7-b65f-431a-8718-255ce455a34d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfStockByClassTable_ad4c23dc-ff82-454a-8d2b-2f8c125c7f7c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfStockByClassTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfStockLineItems_658fbef7-b65f-431a-8718-255ce455a34d" xlink:to="loc_us-gaap_ScheduleOfStockByClassTable_ad4c23dc-ff82-454a-8d2b-2f8c125c7f7c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_f19f9bd3-f40f-43d0-bd5c-32aa24e41a72" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_ad4c23dc-ff82-454a-8d2b-2f8c125c7f7c" xlink:to="loc_us-gaap_PlanNameAxis_f19f9bd3-f40f-43d0-bd5c-32aa24e41a72" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_f19f9bd3-f40f-43d0-bd5c-32aa24e41a72_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_f19f9bd3-f40f-43d0-bd5c-32aa24e41a72" xlink:to="loc_us-gaap_PlanNameDomain_f19f9bd3-f40f-43d0-bd5c-32aa24e41a72_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_14a0a9bd-198f-4b97-9711-16672780a8b2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_f19f9bd3-f40f-43d0-bd5c-32aa24e41a72" xlink:to="loc_us-gaap_PlanNameDomain_14a0a9bd-198f-4b97-9711-16672780a8b2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_A2021EquityIncentivePlanMember_66c8912b-8eff-4a5e-a2c3-cedffa5af286" xlink:href="naut-20250930.xsd#naut_A2021EquityIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_14a0a9bd-198f-4b97-9711-16672780a8b2" xlink:to="loc_naut_A2021EquityIncentivePlanMember_66c8912b-8eff-4a5e-a2c3-cedffa5af286" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_A2021EmployeeStockPurchasePlanMember_39428ac4-ec98-4971-be3a-0ed6515ca1a0" xlink:href="naut-20250930.xsd#naut_A2021EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_14a0a9bd-198f-4b97-9711-16672780a8b2" xlink:to="loc_naut_A2021EmployeeStockPurchasePlanMember_39428ac4-ec98-4971-be3a-0ed6515ca1a0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_c5fbd2e3-1b6d-4349-981e-f5c747199518" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_658fbef7-b65f-431a-8718-255ce455a34d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_c5fbd2e3-1b6d-4349-981e-f5c747199518" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_28f71618-8dcb-467f-957d-fd0b759b5950" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_658fbef7-b65f-431a-8718-255ce455a34d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_28f71618-8dcb-467f-957d-fd0b759b5950" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_ccf40d0e-8f95-452f-aca8-1919b161ea59" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_658fbef7-b65f-431a-8718-255ce455a34d" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_ccf40d0e-8f95-452f-aca8-1919b161ea59" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/EquityIncentivePlansandStockbasedCompensationNarrativeDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#EquityIncentivePlansandStockbasedCompensationNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/EquityIncentivePlansandStockbasedCompensationNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_7e821c7f-5dd3-4e17-a93b-1ade2e90a268" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_7e821c7f-5dd3-4e17-a93b-1ade2e90a268" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_5bd37d53-0e67-4d67-9218-44cdf89b1efa" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_7e821c7f-5dd3-4e17-a93b-1ade2e90a268" xlink:to="loc_us-gaap_AwardTypeAxis_5bd37d53-0e67-4d67-9218-44cdf89b1efa" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_5bd37d53-0e67-4d67-9218-44cdf89b1efa_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_5bd37d53-0e67-4d67-9218-44cdf89b1efa" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_5bd37d53-0e67-4d67-9218-44cdf89b1efa_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_f2901dde-9f1e-4940-bcd7-432de2a5badb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_5bd37d53-0e67-4d67-9218-44cdf89b1efa" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_f2901dde-9f1e-4940-bcd7-432de2a5badb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_31188b40-2200-4081-900d-5b490e7fea9b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_f2901dde-9f1e-4940-bcd7-432de2a5badb" xlink:to="loc_us-gaap_EmployeeStockMember_31188b40-2200-4081-900d-5b490e7fea9b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_0a47b032-4749-4c3d-bf1b-e772cd3459db" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_f2901dde-9f1e-4940-bcd7-432de2a5badb" xlink:to="loc_us-gaap_EmployeeStockOptionMember_0a47b032-4749-4c3d-bf1b-e772cd3459db" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_a7e71cbb-31e6-4b9e-8b55-7f4faafe5062" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_7e821c7f-5dd3-4e17-a93b-1ade2e90a268" xlink:to="loc_us-gaap_PlanNameAxis_a7e71cbb-31e6-4b9e-8b55-7f4faafe5062" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_a7e71cbb-31e6-4b9e-8b55-7f4faafe5062_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_a7e71cbb-31e6-4b9e-8b55-7f4faafe5062" xlink:to="loc_us-gaap_PlanNameDomain_a7e71cbb-31e6-4b9e-8b55-7f4faafe5062_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_f8ff13ef-056b-4116-80f1-3acbc4185fcc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_a7e71cbb-31e6-4b9e-8b55-7f4faafe5062" xlink:to="loc_us-gaap_PlanNameDomain_f8ff13ef-056b-4116-80f1-3acbc4185fcc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_A2021EquityIncentivePlanMember_e65f5aac-e0d5-4638-904d-fa93e72ed93f" xlink:href="naut-20250930.xsd#naut_A2021EquityIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_f8ff13ef-056b-4116-80f1-3acbc4185fcc" xlink:to="loc_naut_A2021EquityIncentivePlanMember_e65f5aac-e0d5-4638-904d-fa93e72ed93f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_A2021EmployeeStockPurchasePlanMember_9b4a64e8-c43e-4f2e-a2e7-8980859966af" xlink:href="naut-20250930.xsd#naut_A2021EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_f8ff13ef-056b-4116-80f1-3acbc4185fcc" xlink:to="loc_naut_A2021EmployeeStockPurchasePlanMember_9b4a64e8-c43e-4f2e-a2e7-8980859966af" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_d34cbfb5-6984-44a4-a632-322a82835e90" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_d34cbfb5-6984-44a4-a632-322a82835e90" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumEmployeeSubscriptionRate_91b04f62-cf4e-4e38-af05-9d60c6ebd58e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumEmployeeSubscriptionRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumEmployeeSubscriptionRate_91b04f62-cf4e-4e38-af05-9d60c6ebd58e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent_93b37650-eeb3-4bd1-8f5e-1465648e54bf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent_93b37650-eeb3-4bd1-8f5e-1465648e54bf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_ShareBasedCompensationArrangementByShareBasedPaymentAwardOfferingPurchasePeriod_89c0675f-339f-48f6-b168-e8aef9c2a694" xlink:href="naut-20250930.xsd#naut_ShareBasedCompensationArrangementByShareBasedPaymentAwardOfferingPurchasePeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_naut_ShareBasedCompensationArrangementByShareBasedPaymentAwardOfferingPurchasePeriod_89c0675f-339f-48f6-b168-e8aef9c2a694" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized_5d1dc7fd-b92a-4c9e-a979-8fa745eb8bb8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized_5d1dc7fd-b92a-4c9e-a979-8fa745eb8bb8" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfOutstandingStockMaximum_52e042e3-2148-43fc-b48c-4b1f1c32cb0d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfOutstandingStockMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfOutstandingStockMaximum_52e042e3-2148-43fc-b48c-4b1f1c32cb0d" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_ShareBasedCompensationArrangementByShareBasedPaymentAwardIncreaseInNumberOfSharesAvailableForGrant_260b6bbb-bb75-43a9-945a-f8ea6365fa15" xlink:href="naut-20250930.xsd#naut_ShareBasedCompensationArrangementByShareBasedPaymentAwardIncreaseInNumberOfSharesAvailableForGrant"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_naut_ShareBasedCompensationArrangementByShareBasedPaymentAwardIncreaseInNumberOfSharesAvailableForGrant_260b6bbb-bb75-43a9-945a-f8ea6365fa15" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_eb5b4c6e-9138-44ad-ad10-443d9acbfac7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_eb5b4c6e-9138-44ad-ad10-443d9acbfac7" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_b360d3af-3285-439e-8613-94e6a4f63e0f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_b360d3af-3285-439e-8613-94e6a4f63e0f" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized_15a38c95-f159-4ceb-9456-2d5429dd1b0d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized_15a38c95-f159-4ceb-9456-2d5429dd1b0d" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_ec02df40-1243-4256-b767-1f37be2bb981" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_8fa6ad35-ca3f-4327-86fd-d02a7af073e8" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_ec02df40-1243-4256-b767-1f37be2bb981" xlink:type="arc" order="10"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/EquityIncentivePlansandStockbasedCompensationScheduleofSignificantAssumptionsDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#EquityIncentivePlansandStockbasedCompensationScheduleofSignificantAssumptionsDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/EquityIncentivePlansandStockbasedCompensationScheduleofSignificantAssumptionsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5786c880-6c26-49db-8f97-4612d11b0ab4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5786c880-6c26-49db-8f97-4612d11b0ab4" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_fc5e61a9-4b47-4296-bf6c-334fc9879460" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5786c880-6c26-49db-8f97-4612d11b0ab4" xlink:to="loc_us-gaap_AwardTypeAxis_fc5e61a9-4b47-4296-bf6c-334fc9879460" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_fc5e61a9-4b47-4296-bf6c-334fc9879460_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_fc5e61a9-4b47-4296-bf6c-334fc9879460" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_fc5e61a9-4b47-4296-bf6c-334fc9879460_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_8dcb6fcb-f4ac-46a2-8d70-83aee1fd55fd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_fc5e61a9-4b47-4296-bf6c-334fc9879460" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_8dcb6fcb-f4ac-46a2-8d70-83aee1fd55fd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_9c74a7c2-11ab-471a-bc42-c19848a5f6d5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_8dcb6fcb-f4ac-46a2-8d70-83aee1fd55fd" xlink:to="loc_us-gaap_EmployeeStockOptionMember_9c74a7c2-11ab-471a-bc42-c19848a5f6d5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_ac34abe1-99ac-4a28-8aa7-5ff090850e09" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5786c880-6c26-49db-8f97-4612d11b0ab4" xlink:to="loc_srt_RangeAxis_ac34abe1-99ac-4a28-8aa7-5ff090850e09" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_ac34abe1-99ac-4a28-8aa7-5ff090850e09_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_ac34abe1-99ac-4a28-8aa7-5ff090850e09" xlink:to="loc_srt_RangeMember_ac34abe1-99ac-4a28-8aa7-5ff090850e09_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_d7e0612a-9537-4d2b-a15e-7f73be3a5286" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_ac34abe1-99ac-4a28-8aa7-5ff090850e09" xlink:to="loc_srt_RangeMember_d7e0612a-9537-4d2b-a15e-7f73be3a5286" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_f3e5d571-cacf-426f-8d7d-0cfd428840d7" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_d7e0612a-9537-4d2b-a15e-7f73be3a5286" xlink:to="loc_srt_MinimumMember_f3e5d571-cacf-426f-8d7d-0cfd428840d7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_50eac7cc-8ead-460e-a1d9-8aa050640c5d" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_d7e0612a-9537-4d2b-a15e-7f73be3a5286" xlink:to="loc_srt_MaximumMember_50eac7cc-8ead-460e-a1d9-8aa050640c5d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_d2f44852-815a-45ea-8286-6ff320967313" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_d2f44852-815a-45ea-8286-6ff320967313" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum_12a44a31-a27e-4bcb-85df-ba892b8f7467" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum_12a44a31-a27e-4bcb-85df-ba892b8f7467" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum_47158f29-3d01-4c79-9deb-c648823a351e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum_47158f29-3d01-4c79-9deb-c648823a351e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_26fe94ae-3ef4-4d5c-81c2-d3ac0193d2c2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_26fe94ae-3ef4-4d5c-81c2-d3ac0193d2c2" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum_e96c89ab-a78f-4d11-a0e6-ab70b342ca9a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum_e96c89ab-a78f-4d11-a0e6-ab70b342ca9a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum_b20358e9-7360-4695-af8a-93fbd3eda2e1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_dc47806b-0f7d-4d33-8dcb-93e24ae04a6b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum_b20358e9-7360-4695-af8a-93fbd3eda2e1" xlink:type="arc" order="5"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/EquityIncentivePlansandStockbasedCompensationScheduleofStockBasedCompensationExpenseDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#EquityIncentivePlansandStockbasedCompensationScheduleofStockBasedCompensationExpenseDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/EquityIncentivePlansandStockbasedCompensationScheduleofStockBasedCompensationExpenseDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_6b1f33b5-e323-40a0-a4c1-976ff1277b4e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_d483948c-f2c0-4a5a-8af1-e066c506857d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_6b1f33b5-e323-40a0-a4c1-976ff1277b4e" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_d483948c-f2c0-4a5a-8af1-e066c506857d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_17937dab-9b72-4162-b056-b4e654278d5f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_d483948c-f2c0-4a5a-8af1-e066c506857d" xlink:to="loc_us-gaap_AwardTypeAxis_17937dab-9b72-4162-b056-b4e654278d5f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_17937dab-9b72-4162-b056-b4e654278d5f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_17937dab-9b72-4162-b056-b4e654278d5f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_17937dab-9b72-4162-b056-b4e654278d5f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_65c6a3d2-4a3c-4c08-b00e-7a10029e5315" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_17937dab-9b72-4162-b056-b4e654278d5f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_65c6a3d2-4a3c-4c08-b00e-7a10029e5315" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_84b63cf4-cf1a-479c-824a-5eae366a98a4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_65c6a3d2-4a3c-4c08-b00e-7a10029e5315" xlink:to="loc_us-gaap_EmployeeStockOptionMember_84b63cf4-cf1a-479c-824a-5eae366a98a4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationAxis_0b10b2dc-aba7-4106-8e50-4a79c6cbbc4a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_d483948c-f2c0-4a5a-8af1-e066c506857d" xlink:to="loc_us-gaap_IncomeStatementLocationAxis_0b10b2dc-aba7-4106-8e50-4a79c6cbbc4a" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_0b10b2dc-aba7-4106-8e50-4a79c6cbbc4a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_0b10b2dc-aba7-4106-8e50-4a79c6cbbc4a" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_0b10b2dc-aba7-4106-8e50-4a79c6cbbc4a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_f667ad54-7d2a-4b39-9d48-a6c6fa96318f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_0b10b2dc-aba7-4106-8e50-4a79c6cbbc4a" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_f667ad54-7d2a-4b39-9d48-a6c6fa96318f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpenseMember_46d00a9a-89a1-4eef-a286-c13b8ac02566" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_f667ad54-7d2a-4b39-9d48-a6c6fa96318f" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpenseMember_46d00a9a-89a1-4eef-a286-c13b8ac02566" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GeneralAndAdministrativeExpenseMember_f6ea61b3-cfe9-441d-9bc3-b47cc3bbd4eb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_f667ad54-7d2a-4b39-9d48-a6c6fa96318f" xlink:to="loc_us-gaap_GeneralAndAdministrativeExpenseMember_f6ea61b3-cfe9-441d-9bc3-b47cc3bbd4eb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_3b62f03e-65ec-4d5e-83b0-7e87ed8d2c31" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_6b1f33b5-e323-40a0-a4c1-976ff1277b4e" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_3b62f03e-65ec-4d5e-83b0-7e87ed8d2c31" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/BasicandDilutedNetLossperShareScheduleofPotentialSharesofCommonStockExcludedfromtheComputationofDilutedNetLossPerShareDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#BasicandDilutedNetLossperShareScheduleofPotentialSharesofCommonStockExcludedfromtheComputationofDilutedNetLossPerShareDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/BasicandDilutedNetLossperShareScheduleofPotentialSharesofCommonStockExcludedfromtheComputationofDilutedNetLossPerShareDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_b2fa6ab2-964b-4ec6-9a02-f85b4bc8557e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_f778236f-fe14-4516-b3d1-9c4e4ec5937d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_b2fa6ab2-964b-4ec6-9a02-f85b4bc8557e" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_f778236f-fe14-4516-b3d1-9c4e4ec5937d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_47d5f1b6-1660-4a20-98d3-ce7b22270a4d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_f778236f-fe14-4516-b3d1-9c4e4ec5937d" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_47d5f1b6-1660-4a20-98d3-ce7b22270a4d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_47d5f1b6-1660-4a20-98d3-ce7b22270a4d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_47d5f1b6-1660-4a20-98d3-ce7b22270a4d" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_47d5f1b6-1660-4a20-98d3-ce7b22270a4d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_ca08525e-6dac-4297-848b-f9ce443d596f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_47d5f1b6-1660-4a20-98d3-ce7b22270a4d" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_ca08525e-6dac-4297-848b-f9ce443d596f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_1129a31d-4c82-4ebd-8179-36a72c31998c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_ca08525e-6dac-4297-848b-f9ce443d596f" xlink:to="loc_us-gaap_EmployeeStockOptionMember_1129a31d-4c82-4ebd-8179-36a72c31998c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_EmployeeStockPurchasePlanMember_dff42040-fa27-4281-89a2-e5877a8a3d48" xlink:href="naut-20250930.xsd#naut_EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_ca08525e-6dac-4297-848b-f9ce443d596f" xlink:to="loc_naut_EmployeeStockPurchasePlanMember_dff42040-fa27-4281-89a2-e5877a8a3d48" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_b6974c1d-86f1-4fa9-8203-e67d89be4447" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_b2fa6ab2-964b-4ec6-9a02-f85b4bc8557e" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_b6974c1d-86f1-4fa9-8203-e67d89be4447" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.nautilus.bio/role/SegmentInformationScheduleofSignificantSegmentExpensesDetails" xlink:type="simple" xlink:href="naut-20250930.xsd#SegmentInformationScheduleofSignificantSegmentExpensesDetails"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/SegmentInformationScheduleofSignificantSegmentExpensesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_4d7d8a0e-0fe0-4300-ae19-50acd7091db0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_d1bdeeee-4f97-4658-a9bd-4332355e74e0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_4d7d8a0e-0fe0-4300-ae19-50acd7091db0" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_d1bdeeee-4f97-4658-a9bd-4332355e74e0" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_f2de86a9-3bd9-42bf-be91-cfdc61f8b837" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_d1bdeeee-4f97-4658-a9bd-4332355e74e0" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_f2de86a9-3bd9-42bf-be91-cfdc61f8b837" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_f2de86a9-3bd9-42bf-be91-cfdc61f8b837_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_f2de86a9-3bd9-42bf-be91-cfdc61f8b837" xlink:to="loc_us-gaap_SegmentDomain_f2de86a9-3bd9-42bf-be91-cfdc61f8b837_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_3ec24ff7-ad99-4b8d-86ea-906b4c6d542d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_f2de86a9-3bd9-42bf-be91-cfdc61f8b837" xlink:to="loc_us-gaap_SegmentDomain_3ec24ff7-ad99-4b8d-86ea-906b4c6d542d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_ReportableSegmentMember_7e9ddf38-1ae7-48c4-8f1e-76c558d06eab" xlink:href="naut-20250930.xsd#naut_ReportableSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_3ec24ff7-ad99-4b8d-86ea-906b4c6d542d" xlink:to="loc_naut_ReportableSegmentMember_7e9ddf38-1ae7-48c4-8f1e-76c558d06eab" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpensesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_4d7d8a0e-0fe0-4300-ae19-50acd7091db0" xlink:to="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LaborAndRelatedExpense_4725d72d-cbe4-48e0-96d2-6cee35c4c5a4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LaborAndRelatedExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:to="loc_us-gaap_LaborAndRelatedExpense_4725d72d-cbe4-48e0-96d2-6cee35c4c5a4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_ProfessionalServicesAndLaboratoryExpenses_cc4d6535-4981-4e73-b188-5b57eb08ce26" xlink:href="naut-20250930.xsd#naut_ProfessionalServicesAndLaboratoryExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:to="loc_naut_ProfessionalServicesAndLaboratoryExpenses_cc4d6535-4981-4e73-b188-5b57eb08ce26" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_naut_FacilityExpenses_d1096daa-4327-41f9-8d5a-cd29c2a0979b" xlink:href="naut-20250930.xsd#naut_FacilityExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:to="loc_naut_FacilityExpenses_d1096daa-4327-41f9-8d5a-cd29c2a0979b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeBenefitsAndShareBasedCompensation_281c9bf8-15a2-4d95-bb0c-60626a31ee78" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeBenefitsAndShareBasedCompensation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:to="loc_us-gaap_EmployeeBenefitsAndShareBasedCompensation_281c9bf8-15a2-4d95-bb0c-60626a31ee78" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingOtherItemAmount_ebe14125-b744-48c7-9c97-46f6dbfbf8c9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingOtherItemAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:to="loc_us-gaap_SegmentReportingOtherItemAmount_ebe14125-b744-48c7-9c97-46f6dbfbf8c9" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpenses_62c23d6b-a58e-4046-9532-aeceea9af206" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_90a87795-bbb0-4cf7-b1ba-6a1f618769ed" xlink:to="loc_us-gaap_OperatingExpenses_62c23d6b-a58e-4046-9532-aeceea9af206" xlink:type="arc" order="6"/>
  </link:definitionLink>
</link:linkbase>
