Pemigatinib (PMB) is a small molecule inhibitor of fibroblast growth factor receptor 1 (FGFR1), FGFR2 and FGFR3. On April 17, 2020, the US Food and Drug Administration granted accelerated approval for PMB for the treatment of adults with previously treated, unresectable metastatic or locally advanced cholangiocarcinoma with a fibroblast growth factor receptor 2 (FGFR2) fusion or other rearrangement. PMB is considered the first targeted treatment for cholangiocarcinoma approved in the US. In this study, in silico prediction of PMB metabolic stability was done using the WhichP450 module of the StarDrop software package. Further, an LC-MS/MS analytical method was developed for PMB quantification in human liver microsomes (HLM) to experimentally assess metabolic stability. PMB and flavopiridol (FVL), used as an internal standard IS, were resolved using an isocratic mobile phase and a C18 stationary phase. The LC-MS/MS method showed linearity in the range of 5 to 500 ng mL⁻¹ in an HLM matrix (R² = 0.9995). The lower limit of quantification (LLOQ) was 5 ng mL⁻¹, indicating sensitivity. The inter- and intra-day accuracy and precision were within a variability of 10, confirming the reproducibility of the method. The measured in vitro half-life and intrinsic clearance of PMB were 27.29 min and 25.40 μL min⁻¹ mg⁻¹, respectively. PMB showed a moderate extraction ratio suggesting good bioavailability. The developed analytical method is the first LC-MS/MS method specific for PMB quantification with application to metabolic stability assessment.