High performance liquid chromatography(HPLC) was developed to detect the hydrolyzed amino acids and free amino acids in grape seeds with phenyl isothiocyanate(PITC) as the derivative reagent. For chromatographic analysis, a Thermo Acclaim^TM 120 C₁₈ column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase consisted of solvent A and B with the flow rate of 1.0 mL/min by gradient elution. Solvent A was sodium acetate buffer solution (pH 6.5) and solvent B was methanol-acetonitrile-water (20∶60∶20, v/v/v). The temperature of column was maintained at 30 ℃. The wavelength was set at 254 nm. The results showed that the R² of 17 kinds of amino acids were more than 0.999 0 in the range of experimental concentration. The average recoveries of hydrolyzed amino acids were 92.0%～104.6%, and RSDs were 0.83%～4.07%. The average recoveries of free amino acids were 95.2%～102.4%, and RSDs were 1.06%～3.98%. The contents of hydrolyzed amino acids in 6 kinds of grape seeds were 130.46～163.24 mg/g. The contents of free amino acids were 2.04～5.13 mg/g. More free amino acids were contained in the grape seeds from Leisiling, Saimeirong and Guirenxiang which three kinds of grape seeds were usually used to brew the white wine. The contents of hydrolyzed amino acids were higher in the grape seeds from Heipinuo, Shelongzhu and Chixiazhu which were always used for the red wine brewing. The method was stable and reliable, can be used for the determination of hydrolyzed amino acids and free amino acids in grape seeds.