Abstract: The method for simultaneous quantitative determination of Rotundine and Sulpiride in human plasma was established by HPLC-MS/MS. Verapamil was chosen as internal standard. Sample of plasma was alkalified by sodium hydroxide, then extracted with dichloromethane. Rotundine （m/z 356.4/340.4） and Sulpiride（m/z 342.3/214.1）were detected by LC-MS/MS with multiple reaction monitoring mode. The standard curve of Rotundine and Sulpiride are linear in the concentration range of 0.781 3-400 μg•L^-1 (r=0.999 3) and 3.125-1 600 μg•L^-1 (r=0.999 3)， respectively. The method recovery of Rotundine and Sulpiride are 95.3 %-100.8 % and 98.6%102.4%. The intraday RSD are 2.78%-6.79%and 1.56%-2.63%. The inter-day RSD are 0.32%-2.22% and 0.80%-1.41%. This method is simple, rapid, sensitive and accurate for determination of Rotundine and Sulpiride simultaneously. It can be used in pharmacokinetic for studying Rotundine and Sulpiride.