Objective To develop a RP-HPLC method for the determination of Brucine and Strychnine in Semen Strychni and its extractive of total alkaloids. Methods A chromatographic column of Licrospher C18 (4.6 mm×250 mm, 5 μm) was used with the mobile phase of acetonitrile∶0.01 mol/L sodium heptane sulfonate and 0.02 mol/L potassium dihydrogen phosphate mixed with equal amount (adjusted pH to 2.8 with 10% phosphonic acid)=27∶73, detection wavelength at 260 nm, column temperature of 30 ℃ and flow rate of 1 mL/min. Results The calibration curves of Brucine and Strychnine were both in good linearity in the ranges of 0.1-1.0 μg and 0.12-1.2 μg (r=1.000) respectively. The average recovery rates of Brucine and Strychnine were 99.88% (RSD=1.06%) and 100.06% (RSD=0.78%) respectively. Conclusion The method is realiable and accurate, which can be applied to determination of Brucine and Strychnine in Semen Strychi and its extractive.