ABSTRACT Rationale Resigratinib, a potent fibroblast growth factor receptor (FGFR) inhibitor, is under clinical development for solid tumors such as cholangiocarcinoma. However, data on its hepatic metabolism remain limited. To support further development, this study aimed to characterize its in vitro metabolism using rat, dog, monkey, and human liver microsomes. Methods A sensitive and robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was validated for quantifying resigratinib in liver microsomes. Metabolite characterization was performed using LC coupled with benchtop Orbitrap high‐resolution mass spectrometry (LC‐Orbitrap‐HRMS) in full‐scan MS/dd‐MS 2 and parallel reaction monitoring (PRM). This approach enabled accurate mass measurement, chemical formula assignment, and structural elucidation via MS 2 fragmentation interpretation. Results The established method exhibited excellent linearity over the concentration range of 1.0–1000 nM. Resigratinib displayed low clearance in dog ( t 1/2 = 91.2 min), intermediate clearance in rat ( t 1/2 = 20.2 min), and high clearance in monkey ( t 1/2 = 6.8 min) and human ( t 1/2 = 14.0 min) systems. Ten metabolites were identified, with M3 ( bis ‐demethylation), M5 ( O ‐demethylation), and M9 ( N ‐demethylation) identified as the major metabolites. Recombinant human cytochrome P450 enzyme analysis and chemical inhibition studies indicated that CYP3A4 is the predominant enzyme responsible for resigratinib metabolism. Conclusion This study presents the first integrated analytical approach, combining LC–MS/MS and LC‐Orbitrap‐HRMS, for the in vitro metabolic assessment of resigratinib. The observed metabolic profiles provide an essential foundation for further toxicological and clinical investigations.