Enzymatic hydrolysis of shark (Squatina oculata) cartilage (SC) was optimized by response surface methodology (RSM) for chondroitin sulfate (CS) preparation. Among 11 commercial proteases, Maxazyme NNP showed highest productivity (CS yield per enzyme cost) of CS. Optimal hydrolysis conditions determined by RSM were 1.63% and 2.87 hr for enzyme concentration and hydrolysis time ($r^2;=;0.9527$, p<0.0l), respectively and highest yield of hydrolysate under the conditions was 42.3%. The yield ($43.1{pm}2.1%$) and CS content ($24.8{pm}0.1%$) of hydrolysate at optimal condition verified statistical optimization of SC enzymatic hydrolysis was valid.