BPI-9016M is a novel dual-target small-molecule inhibitor targeting c-Met and AXL, which was developed by Betta Pharmaceuticals Co., Ltd (Hangzhou, China). It has great potential in the treatment of advanced cancer. A high throughput quantitation method, based on liquid chromatography-tandem mass spectrometry, was developed and validated for the simultaneous determination of BPI-9016M and its main metabolite, M1 and M2-2, in human plasma with a sample preparation method of precipitation of protein. Liquid chromatographic separation was performed with a gradient elution of formic acid-10mM ammonium acetate aqueous solution (1:1000, v/v) and acetonitrile at a flow rate of 0.4mL/min within 2.2min. A Waters ACQUITY UPLC BEH C18 column (1.7μm, 2.1×50mm) was chosen, of which the temperature was set to be 40°C. Mass spectrometric detection, which were achieved in positive mode, were performed by multiple reaction monitoring with SCIEX API 5500 Qtrap equipped with an ESI ion source. This method showed good linearity, accuracy and precision in the range of 0.4-200ng/mL for BPI-9016M and 0.8-800ng/mL for M1 and M2-2, with high recovery and slight matrix effect for all analytes. And under the conditions same as stability assessments in method validation, the three analytes stayed stable during the entire destiny of a clinical sample from the collection of whole blood to the analysis of plasma by this method. The validated method was successfully applied to a first-in-human, dose-escalation phase I clinical trial in Chinese advanced solid tumor patients for the pharmacokinetic research of BPI-9016M tablet after oral administration. The concentration-time curves of BPI-9016M, M1, M2-2 were detailly captured with good veracity. And according to the results of hemolysis assessment, plasma concentrations of analytes in hemolyzed plasma samples could be reported normally without label.