Abstract: OBJECTIVE To establish an HPLC method for determination of plasma concentration of leonurine (LE) and investigate its pharmacokinetics in rats. METHODS After oral administration of LE suspension (50 mg·kg^-1), plasma samples were collected at different points. After extracted from plasma by ethyl acetate, the plasma concentrations of LE and its internal standard (IS) n-benzoyl-L-arginine ethyl ester (BAEE) were determined by HPLC. Chromatographic separation was performed on a Diamonsil C₁₈(250 mm×4.6 mm, 5 μm) with UV detection at 277 nm, using acetonitrile-0.02 mol·L^-1 monopotassium phosphate (pH 3.0) (22:78) as mobile phase at 1.0 mL·min^-1 flow rate, and the column temperature was 35℃. The pharmacokinetic parameters were calculated by PKS 1.0 software program. RESULTS The linear calibration curve of LE was obtained in the concentration range of 0.05-1.5 μg·mL^-1(r=0.999 1), and the lower limit of quantitation(LLOQ) was 0.05 μg·mL^-1(RSD=12.8%, n=5); The absolute recovery in plasma was 76.5%-82.5%; Intra-day and inter-day relative standard deviations were both below 10%, with accuracy in the range 96.9%-104.9%. The QC plasma samples were stable through repeated three freeze/thaw cycles and under the frozen condition at -20℃ for 30 d. The process of LE in rat fit two-compartment open model, and the main pharmacokinetic parameters obtained were t_max=0.95 h, C_max=0.51 μg·mL^-1, t_1/2=3.64 h, AUC_0-t=1.56 μg·mL^-1·h^-1, AUC_0-∞=1.78 μg·mL^-1·h^-1. CONCLUSION The assay method is proved to be simple, sensitive, precise and reliable enough for the pharmacokinetics study of LE in rat after oral administration.