Protein hydrolysate was prepared from common cuttlefish Sepia officinalis by Pepsin. Hydrolysis conditions (time, temperature, and enzyme activity) were optimized by response surface methodology (RSM) using a factorial design. The regression coefficient was close to 0.999, observed during both experimental and validation runs, and indicated the validity of prediction model. An enzyme activity to substrate ratio (X1), 0.04 AU/g protein; time (X2), 85 minutes; and temperature (X3), 45 °C were found to be the optimum conditions for a higher degree of hydrolysis (21%) and nitrogen recovery (90%) using Pepsin. According to amino acid analysis results and chemical score, methionine and histidine are the limiting amino acids in the hydrolysates, in that order. All other amino acids are present in sufficient quantity as required.