Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is critically involved with cell migration, distributing and proliferation in the early step of numerous cancers. Small molecule inhibitors of FAK work well to hinder its activation while tumor formation in cell. To higher understand biotransformation of FAK inhibitors, the work has investigated in vitro phase I metabolic process of inhibitors (namely PF-573228, PF-562271 and PF-03814735) by rat liver microsomes model. Using liquid chromatography – quadrupole duration of flight mass spectrometry and tandem mass spectrometry (LC/Q-TOF/MS and MS/MS), three metabolites of PF-573228 and PF-562271 were observed and characterised, correspondingly. These in vitro metabolites were reported the very first time. The structures and fragmentation patterns of those metabolites were elucidated, and phase I metabolic pathways for FAK inhibitors were suggested. The primary metabolic pathways of PF-573228 were hydroxylation, dehydrogenation and N-dealkylation. For PF-562271, these were hydroxylation and dehydrogenation. Hydroxylation was observed because the primary metabolic process for PF-0381473.