OBJECTIVE：To improve the content determination for polygala xanthone Ⅲ in Polygalae Radix contained in Chinese Pharmacopoeia（2010 edition）. METHODS：HPLC was performed on the column of Hypersil BDS C18 with mobile phase of acetonitrile-0.05％ phosphoric acid（gradient elution）at a flow rate of 1.0 ml/min；detection wavelength was 320 nm，column temperature was 25 ℃，and the injection volume was 10 μl. RESULTS：The linear range of polygala xanthone Ⅲ was 0.029-0.928 μg/ml （r＝0.999 1）；RSDs of precision，stability and reproducibility tests were lower than 2.0％；recovery was 94.66％-100.90％（RSD＝ 2.46％，n＝6）. CONCLUSIONS：The method is simple，stable and reproducible. Although the determination time is prolonged，it has improved the accuracy and it is more suitable for the content determination of polygala xanthone Ⅲ in Polygalae Radix.