Gelatin hydrolysate is a product with a wide range of benefits in pharmaceutical and food applications. Therefore, this study analyzes the best hydrolysis condition to make Barramundi (Lates calcarifer) scales gelatin hydrolysate using ultrasonication and lime juice. The conditions with the best degree of hydrolysis (DH) were optimized using the Response Surface Method (RSM) with Box Behnken Design (BBD). The effects of 3 independent variables, namely time, temperature, and enzyme concentrations, were then examined for model optimization. In addition, the optimized hydrolysis was performed at 31.03 mins, 56.84оC temperature, 1.5% E/S concentration, and pH 8.0. At these optimal conditions, alkaline protease hydrolysis yielded a 1.000 desirability value, with a 64.83% DH. Amino acid (AA) with the highest value in Barramundi scales gelatin hydrolysate was lysine (53.03 g/100 g), while isoleucine (0.05 g/100 g) had the lowest value. Based on the results, optimal hydrolysis conditions could be an excellent reference to increase the potential of Barramundi scales gelatin hydrolysate in developing future functional foods with high amino acids, particularly lysine.