Abstract: Objective To establish a high-performance liquid chromatography (HPLC) for determining stachydrine and leonurine contents in the crude drug of Yimu Cao, a Chinese motherwort herb(Herba Leonuri, the aerial part of Leonurus japonicus).Methods The sample was obtained from the crude drug by ultrasonic extraction with ethanol after decolorization with ethyl acetate. YMC-Park CN column (250.0 mm×4.6 mm) was used with the mobile phase for stachydrine of 0.001 25 mol/L SDS (containing 0.05% HClO₄):C2H5CN (90:10) at the flow rate of 1.5 ml/min with the wavelength for detection of 201.7 nm, and that for leonurine of 0.00125 mol/L SDS (containing 0.05% HClO₄):CH3OH (90:10) at the flow rate of 1 ml/min with the wavelength for detection of 282 nm. The sensitivity was 0.1 AUFs and the column temperature was 20℃.Results With the injection amount (mg) as the abscissa and the peak area as the ordinate, the regression equation of the calibration curve for stachydrine was Y=1 187.542 3X-168.982 2, and that for leonurine was Y=5 202.654X-221.141 (r=0.9998) with the linearity scope of 2.5-12.5 mg, detective limit of 0.15 mg, and recovery of (99.03±2.744)%. The analysis of extracted drug samples from 18 regions indicated that the alkaloids contents in Yimu Cao varied significantly, i.e. stachydrine within a range of 0.1%-0.2% while leonurine content, which was much lower, within 0.01%-0.05%. In general, the alkaloid contents were higher in the drug produced in northern China than in those produced in southern China. L. japonicus and the variant L. japonicus albiflorus had total alkaloid contents around 0.3%, which was higher than the contents of other species.Conclusion As a convenient and feasible means for determining the alkaloid contents in Yimu Cao, HPLC produces accurate and reliable results and can be more effective than the formerly used methods.