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A simple, sensitive and selective method of high-performance liquid chromatography (HPLC) has been successfully developed for separation of bavachinin enantiomers in Fructus Psoraleae and rat plasma. The separation and detection conditions of HPLC were optimized. Chiral bavachinin were separated with the mobile phase of methanol and water (70:30, v/v) at a flow rate of 1.0mL/min. The linear ranges were in the range of 20â1000μg/mL. The detection limits were tested as 4ng/mL and 6ng/mL for (+)-bavachinin and (â)-bavachinin, respectively. The method has been applied to analyze chiral bavachinin in rat plasma. HPLCâMS method was used to test the accuracy. Keywords: Bavachinin, Fructus Psoraleae, HPLCâMS, HPLC-UV, Rat plasma