Objective To develop a qualitative and quantitative simultaneous determination of multiple bioactive constituents in total alkaloid in Sophora alopecuroides (TASA). Methods In the experiment, a new and simple HPLC-DAD method for the simultaneous determination of multiple constituents in TASA was developed. The separation was performed on a Kromasil C,8 column (250mmx4.6mm,5.0pm) eluted with 0.02 mol/L potassium dihydrogen phosphate (adjusted pH 4.3 using 1% glacial acetic acid) and acetonitrile (75:25) at a flow-rate of 0.7 mUmin. The detection wavelength was set at 210 nn. Results Five constituents (sophoridine, matrine, oxymatnne, aloperme, and lehmamune) were simultaneously analyzed in this study. Four of them were identified and determinated by the developed method. The calibration curves exhibited linear regressions (r~2>0.9995). The injection precision, the intra-day precision, and the ana hsis repeatability were validated with the RSD values less than 5.0%. The mean recoveries of the four constituents were ranged from 98.62% to 100.20%, and the RSD values were all less than 3.37%. Conclusion This method is convenient, fast, accurate, and is applicable to analyze the multi-constituents in TASA.