An HPLC method was used for determination of cholic acid and hyodeoxycholic acid.Using HPLC-UV with Diamonsil-C18 column (4.6 mm × 150 mm, 5 μm), acetonitrile-water-phosphoric acid (35:65:0.1) as the mobile phase; the detection wavelength was 192 nm; the temperature of the column was 25°; the flow rate was 1.5 mL/min-1.A better linearity and good correlation coefficient were obtained: cholic acid was 3.154-31.540 μg, r = 0.999 5; hyodeoxycholic acid was 1.927-19.266 μg, r = 0.9992.The satisfactory reconvenes were obtained: cholic acid was 98.98%, RSD = 0.42%; hyodeoxycholic acid was 98.29%, RSD = 0.61%.The method was accurate, reliable, and repeatable, which can be used as the method of quality control for cholic acid and hyodeoxycholic acid.