A sensitive, accurate and rapid high-performance liquid chromatography with UV-visible detection (HPLC-UV) method for the determination of Gemfibrozil in human plasma has been developed. Gemfibrozil and internal standard (Naproxen) were extracted by using solid-phase extraction technique with direct elution. The resolution of peaks was achieved with 10 mM potassium dihydrogen phosphate buffer (pH 4.0 + 0.1) and acetonitrile in a ratio of 95:5 v/v on a reversed-phase X-Terra C18 column (4.6 mm Internal Diameter X 150 mm Length, 5μ particle size). The eluents were quantified by UV detection at 222 nm. The analytical run time was less than 12.5 min. The method was proved to accurate and precise at linearity range of 0.202–50.070 μg/mL. The method was applied to a pharmacokinetic study and bioequivalence study of gemfibrozil.