Abstract: To develop a sensitive and specific high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of mosapride in human plasma, mosapride and internal standard tamsulosin were extracted from plasma with liquid-liquid extraction, then separated on a Waters ACQUITY UPLC^TM BEH C₁₈ column (50 mm×2.1 mm, 1.7 μm ID) with gradient elution at flow-rate of 0.25 mL·min^-1. The mobile phase was water (containing 0.3% formic acid) and acetonitrile under gradient conditions. Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 422 → m/z 198 and m/z 409 → m/z 228 were used to quantify mosapride and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 0.17-68.00 ng·mL^-1. The lower limit of quantification was 0.17 ng·mL^-1. The inter- and intra-day precision (RSD) was less than 13%, and the accuracy (RE) was within ±6.3% calculated from QC samples. The method was used to determine the concentration of mosapride in plasma after a single oral dose of 5 mg mosapride citrate to 20 healthy male Chinese volunteers. The method has been proved to be selective, sensitive, rapid and suitable for pharmacokinetic study of mosapride.