In this study the viscera of Pangasiidae discarded from fillet processing was hydrolyzed by alcalase 2.4L in order to obtain protein hydrolysate with high calcium-binding activity. Hydrolysis conditions (enzyme activity, temperature, and time) were optimized using response surface methodology to maximize the calcium-binding activity of hydrolysate. The optimum conditions to reach the highest calcium-binding activity were: 67oC, 130 min, and enzyme to substrate ratio of 0.3 percent (v/w). Degree of hydrolysis of the optimum hydrolysate was 15 percent. In addition, result of SDS-PAGE gel electrophoresis indicated that 90 percent peptides of the optimum protein hydrolysate had molecule weight of under 3.4 kDa. Therefore, these results suggested that the small peptides derived from Pangasiidae processing byproducts protein hydrolysates is responsible for calcium binding properties.