Abstract: A high performance liquid chromatography with fluorescence detection has been developed for determination of puerarin in plasma using a column of YWG-C₁₆ (10 μm) and mobile phase of methanol (450 ml)-water (522.5 ml)-0.1 mol·L^-1 phosphate buffer (pH 7.4, 27.5 ml). The preparation of samples was as follows: Transfer 0.1 ml of plasma into a 1 ml stoppered glass tube containing daidzein as internal standard, add 0.4 ml of absolute alcohol to deproteinize the plasma, vortex for 2 min, centrifuge for 15 min at 2800 r·min^-1 and inject 20 μl of the supernatant into the HPLC column. Linear calibration curve was obtained by plotting concentration vs peak height ratio over the range 0.03～12.00 μg·ml^-1 with a correlation coefficient of 0.999. The average recovery of puerarin was 95.3% and the average RSD was 4.8%. The minimum detectable quantity of puerarin was 0.04 ng, equivalent to 10 ng·ml^-1 plasma. This method is highly sensitive, specific, simple and rapid. The plasma concentrationtime course of puerarin given iv 2.25 mg·kg^-1 to two dogs determined by this method was found to fit a two-compartments open model with T_1/2α of 6.0 min and T_1/2β of 57.4 min.