Abstract: A new analytical method for the separation and simultaneous determination of three saponins in Licorice root by high performance liquid chromatography has been developed. The saponins, glycyrrhizic acid (S-I), uralsaponin B (S-Ⅱ) and uralsaponin A (S-Ⅲ) were separated and determined by use of the solvent system CH₃CN—3% HOAc (H₂O)(47: 53) at 248nm, and with gradient increasing flow rate, the operation can be completed in 20min. This method is sensitive and acurate with good reproducibility. The contents of three saponins in Chinese Licorice roots derived from four Glycyrrhiza species were determined.