A method for determination of cinobufagin and recibufogenin in Chanshe Jiuxin pills by RP-HPLC was established.Kromasil C18 column was used, with acetonitrile-water (0-25 min, 56:44; 26-44 min, 0:100; 45-50 min, 56:44, volume/volume) gradient elution as a mobile phase at a flow rate of 1.0 mL/min.The wavelength of detection was 250 nm.The column temperature was 35°.The determination of cinobufagin showed a good linear relationship in the range of 0.2118-2.118 μg (r = 0.9997), with the average recovery of cinobufagin 99.49% and RSD 1.2% (n = 6).The determination of recibufogenin showed a good linear relationship in the range of 0.2012-2.012 μg (r = 0.9996).The average recovery of recibufogenin was 99.97%, and RSD was 1.7% (n = 6).The method is simple, accurate, and precise, which can be used for the assay of Chanshe Jiuxin pills.