OBJECTIVE：To establish a method for the content determination of glabridin in Clycyrrhizae Radix et Rhizoma from different regions. METHODS：HPLC was performed on the column of Inertsil ODS-SP with mobile phase of acetonitrile-0.05％ phosphoric acid （gradient elution） at a flow rate of 0.8 ml/min， detection wavelength was 280 nm， the column temperature was 35 ℃， and the injection volume was 20 μl. RESULTS： The linear range of glabridin was 0.906-18.12 μg/ml（r＝0.999 7）， RSDs of precision， stability and reproducibility tests were lower than 3％； recovery was 98.73％-101.90％（RSD＝1.25％，n＝6）. There were obvious differences among the glabridin contents in G. uralensis from different regions. CONCLUSIONS：The method is simple and accurate with high precision， and can be used for the content determination of glabridin in Clycyrrhizae Radix et Rhizoma.