ABSTRACTCanertinib is an EGFR tyrosine kinase inhibitor intended for the treatment of leukemia and non–small cell lung cancer. This study described a UPLC‐MS/MS method for quantitatively assessing the metabolic stability of canertinib in liver microsomes. The developed method showed excellent linearity over the concentration range of 10–1000 nM, which is suitable for in vitro high‐throughput screening. Canertinib showed marked species‐dependent metabolism, with CLin vitro, mic following the order: human (28.3 μL/min/mg protein) < rat (48.2 μL/min/mg protein) < monkey (77.8 μL/min/mg protein). An LC‐Orbitrap‐HRMS facilitated structural characterization of the metabolites via accurate mass measurements and MS/MS fragmentation interpretation. Post‐acquisition data‐mining strategies, specifically high‐resolution extracted ion chromatograms and multimass defect filtering, were employed to screen the putative metabolite candidates. Sixteen NADPH‐dependent metabolites and one GSH conjugate were structurally characterized. Cross‐species comparative analysis revealed notable interspecies variations: metabolites M9 and M17 were identified as human‐specific, while M15 and M16 demonstrated monkey‐specificity. The metabolic pathways of canertinib included oxidative defluorination, O‐dealkylation, oxidative deamination, piperidine ring opening, lactam formation, and GSH conjugation. This work represents the first cross‐species metabolic investigation of canertinib, providing critical insights into interspecies metabolic disparities. The elucidated metabolic framework advances mechanistic understanding of the compound's pharmacological activity and toxicity profiles.