ABSTRACT Rationale Sitravatinib is a receptor tyrosine kinase inhibitor that has been developed for the treatment of advanced nonsmall‐cell lung cancer (NSCLC) and urothelial carcinoma. This study aimed at developing an integrated LC–MS/MS and LC–Orbitrap–HRMS platform for evaluating the metabolic stability and profiling the metabolites in rat and human liver microsomes. Method Separation was achieved using a Waters ACQUITY BEH C 18 column with a gradient of 0.1% formic acid in water and acetonitrile. Detection utilized positive electrospray ionization and multiple reaction monitoring of transitions m/z 630.2 → 555.2 for sitravatinib and m/z 502.5 → 323.2 for the internal standard. Metabolite identification was performed using LC–Orbitrap–HRMS through full‐scan MS/dd‐MS 2 and parallel reaction monitoring. The structures of the metabolites were characterized via accurate mass measurement and MS 2 fragmentation interpretation. Results The LC–MS/MS method showed excellent linearity over a concentration range of 1.0–2000 nM, suitable for high‐throughput in vitro assays. Sitravatinib demonstrated poor metabolic stability ( t 1/2 = 17.47 ± 2.57 min) in rat liver microsomes, with seven metabolites identified. Among these, M3 and M5 were the major metabolites. In contrast, sitravatinib exhibited high metabolic stability in human liver microsomes ( t 1/2 = 96.06 ± 12.17 min), with only seven minor metabolites detected. Conclusions Key metabolic pathways included O ‐demethylation, amide formation, N ‐dealkylation, and oxidative deamination. This study establishes the first integrated LC–MS/MS and HRMS strategy for in vitro metabolic profiling of sitravatinib.