Sunitinib,　N-desmethyl　imatinib,　dasatinib,　imatinib,　and　bosutinib　are　tyrosine　kinase　inhibitors　(TKIs)　that　are　commonly　employed　in　the　treatment　of　a　multitude　of　cancers.　However,　the　inappropriate　concentrations　of　TKIs　can　result　in　ineffective　treatment　or　the　emergence　of　multiple　adverse　effects.　Consequently,　the　development　of　a　rapid　and　sensitive　analytical　method　for　TKIs　is　of　paramount　importance　for　the　safe　administration　of　drugs.　In　this　work,　solid-phase　microextraction　(SPME)　probe　combined　with　an　electrospray　ionization　mass　spectrometry　(ESI-MS)　coupling　platform　was　constructed　for　rapid　and　sensitive　determination　of　TKIs.　The　covalent　organic　frameworks　(COFs)　coated　SPME　probe　was　made　of　2,4,6-tris(4-aminophenyl)-1,3,5-triazine　(TAPT)　and　2,5-dibutoxyterephthalaldehyde　(DBTA)　by　in-situ　layer-by-layer　chemical　bonding　synthesis　strategy.　The　TAPT-DBTA-SPME　probe　exhibited　several　advantageous　properties　which　rendered　it　suitable　for　the　enrichment　of　TKIs.　Under　the　optimal　conditions,　the　developed　analytical　method　demonstrated　a　broad　linear　range　(0.05–500.00　µg/L),　a　low　limit　of　detection　(0.02　µg/L)　and　a　high　enrichment　factor　(51–203)　for　TKIs.　The　developed　analytical　method　was　successfully　applied　to　a　pharmacokinetic　study　of　TKIs　in　mouse　plasma　and　tissue　matrix,　demonstrating　that　the　proposed　analytical　method　has　promise　for　clinical　applications　and　metabolic　monitoring.　©　2024　Elsevier　B.V.