The purpose of the paper was to determine quant. cinobufagin and resibufogenin for chief component of venenum bufonis in Liushen pill by HPLC.The column was Hypersil C18 (250 mm × 4.6 mm, 5 μm), and the mobile phase was acetonitrile-water (43:57, V/V).The flow rate was 1.0 mL/min-1, and the detection wavelength was 296 nm with column temperature of 25°.Results showed that the linear range of cinobufagin was 0.0525-1.050 μg (r = 0.999), and the average recovery was 100.18% (RSD = 1.85%; n = 6).The linear range of resibufogenin was 0.052-1.040 μg (r = 0.999), and the average recovery was 100.40% (RSD = 1.14%; n = 6).In conclusion, the method was simple, rapid, and accurate, and could be applied to the determination of cinobufagin and resibufogenin in Liushen pill.