Abstract: The purpose was to establish a method for quantitative determination angiotensin converting enzyme (ACE) inhibitory peptides in hydrolyzate of casein using reversed-phase high performance liquid chromatography ( RP-HPLC) . Casein was hydrolyzed by pepsin, and then was hydrolyzed by trypsin. With flavourzyme for debittering of casein hydrolyzate. Salts of casein hydrolyzate were removed by ion exchange resin. The RP-HPLC method to detect decapeptide YQKFPQYLQY was established. Using external standard method to determine YQKFPQYLQY of casein hydrolyzate. Results showed that, the linear range of decapeptide was 2.5⁴.5mg/m L (R2=0.95496) . The decapeptide concentration in the casein hydrolyzate was 4.96694%±0.00131%.Therefore, the RP-HPLC was simple and rapid, suitable for the quantitative detection of ACE inhibitory peptides YQKFPQYLQY.