A simple and specific high-performance liquid chromatographic (HPLC) method was developed for the pharmacokinetic study of glibenclamide in rats after oral administration of XiaoKe pill. The plasma samples were deproteinized with acetonitrile after addition of internal standard (i.s.) glipizide. HPLC analysis was performed on a SinoChrom ODS-BP C18 analytical column, using acetonitrile - 0.1% acetic acid (44:56, v/v) as the mobile phase with UV detection at 230 nm. The standard curve was linear over the range of 10.2 - 306.0 ng/mL in rat plasma. This method is successfully applied for the pharmacokinetic study of glibenclamide in rat after oral administration of three doses of XiaoKe pill.