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Glycosaminoglycan (GAGs) is one kind of mucopolysaccharide, the natural biocompound can be extracted from cartilage of sharks, pigs, cows, chickens, etc. It contains chondroitin‐4‐sulphate, chondroitin‐6‐sulphate, heparansulphate, dermatansulphate, keratan sulphate, and hyaluronic acid, links with protein chains and thus are called proteoglycan. Chondroitin‐4‐sulfate (CS), the highest proportion in GAGs of poultry cartilage, is using as a supplement of functional food for preventing and supporting treatment of arthritis and eye tissues. Therefore, it is necessary to release the CS from chicken cartilage that is abundant by‐products of poultry processing. In this study, the hydrolysis conditions by protamex of chicken cartilage were investigated. The variables such as: pH, protamex content, incubation temperature, and hydrolysis time were optimized. The results of the analysis of variance (ANOVA) shown that the variables actively affected CS content. The optimal conditions of hydrolysis were pH of 5.81, ratio of enzyme per substances of 0.85 % w/wprotein, temperature of 54.63°C and hydrolysis time of 204 minutes, GAGs content reached 25.28 %. Analysis by HPLC revealed CS content 17.69 % dry matter of raw material