MRTX849 is a novel, highly selective, targeted inhibitor of KRAS (G12C), which significantly improves the objective response rate in patients with advanced solid tumors. However, neither an analytical HPLC-MS/MS assay nor pharmacokinetics has been reported for MRTX849 in plasma. In the present study, chromatography was accomplished on a reversed phase C18 column (50 × 2.1 mm, 3.5 μm). The limit of detection of MRTX849 was 0.02 ng mL-1 at S/N ≥ 3. Only 20 μL of plasma was utilized for accurate quantitation. The optimized analytical assay was fully validated and verified in accordance with guidelines. The calibration curve for MRTX849 was linear with a correlation coefficient >0.99 in the range of 0.05-200 ng mL-1. The intra- and inter-day accuracy and precision were all within ±10%. The matrix effect and recovery were consistent and acceptable under several quality control concentrations. This HPLC-MS/MS method was successfully applied for a pharmacokinetic study of MRTX849 at a dose of 15 mg kg-1.