Aims: This study aimed at developing bioactive peptides by optimization of the enzymatic hydrolysis process of protein from tilapia fish skin waste (Oreochromis niloticus, Linnaeus 1758) using alcalase 2.4.L. Background: Natural bioactive peptides are considered to have low toxicity and therapeutic properties as antioxidants. Objective: The conditions of protein hydrolysis obtained from tilapia fish skin waste (Oreochromis niloticus, Linnaeus 1758) were optimized using alcalase 2.4.l Methods: In this study, the hydrolysis of protein obtained from tilapia fish skin waste (TFSW) was optimized using alcalase 2.4.L by central composite design (CCD). Degree of hydrolysis (DH), radical scavenging activities (DPPH), and ferric-reducing antioxidant power (FRAP) were used as dependent variables, whereas temperature, pH, and proportion of enzyme to the substrate (PE%) as independent variables. Results: The optimum degree of hydrolysis DH%, DPPH, and FRAP were achieved at a temperature of 58.4 ℃, a pH of 8.7, except for DPPH, which was achieved at a pH of 7.0. Conclusion: The present work demonstrated that TFSW could be used as a source to produce bioactive peptides with significant antioxidant activities under specific conditions of enzymatic hydrolysis.