Bruton's tyrosine kinase (BTK) inhibitors play a critical role in the treatment of mantle cell lymphoma (MCL). pirtobrutinib, a new, highly selective, non-covalent BTK inhibitor, was approved by the FDA for the treatment of MCL, chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL). In this study, we established a robust and reliable method for the quantitation of pirtobrutinib in rat plasma using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Acetonitrile and 0.1% formic acid served as the mobile phase, with zanubrutinib as the internal standard (IS). Detection ion transitions were m/z 480.12→294.05 for pirtobrutinib and m/z 472.20→ 289.96 for Zanubrutinib. The intra-day and inter-day relative standard deviation (RSD%) values of pirtobrutinib were less than 9.8% and 10.3%, respectively. Recovery and matrix effects ranged from 95.1 to 101.5% and 91.7-100.4%. In addition, the test sample stability was confirmed under various storage conditions, and this method was successfully applied to a pharmacokinetic study of pirtobrutinib at a dose of 10 mg·kg