This study was conducted to identify and characterize antioxidant peptides from the alcalase hydrolysate of the blue-spotted stingray. Purification steps guided by ABTS cation radical (ABTS ) scavenging assay and de novo peptide sequencing produced two peptides, WAFAPA (661.3224 Da) and MYPGLA (650.3098 Da). WAFAPA (EC = 12.6 µM) had stronger antioxidant activity than glutathione (EC = 13.7 µM) and MYPGLA (EC = 19.8 µM). Synergism between WAFAPA and MYPGLA was detected. WAFAPA and MYPGLA surpassed carnosine in their ability to suppress H O -induced lipid oxidation. The peptides protected plasmid DNA and proteins from Fenton's reagent-induced oxidative damage. Thermal (25–100 °C) and pH 3–11 treatments did not alter antioxidant activity of the peptides. MYPGLA maintained its antioxidant activity after simulated gastrointestinal digestion, whereas WAFAPA showed a partial loss. The two peptides may have potential applications as functional food ingredients or nutraceuticals, whether used singly or in combination.