Selpercatinib (RETEVMO®) is a selective anticancer agent recently approved for thyroid and non-small cell lung cancer. Reliable analytical methods are essential for investigating its potential drug interactions. In this study, the fluorescence properties of selpercatinib were exploited for the first time to develop a sensitive high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method to quantify selpercatinib in human and rat liver microsomes and rat plasma. The method was successfully validated according to M10 guidelines demonstrating excellent accuracy, precision, selectivity, and sensitivity across a concentration range of (50-2000) ng/mL in plasma samples, with a short run time of less than 4 min. The method was applied to metabolic stability studies, where selpercatinib exhibited moderate intrinsic clearance (CL