To utilize fish processing waste, tuna backbone protein was hydrolyzed using different proteases (alcalase, alpha-chymotrypsin, neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, peptic hydrolysate exhibited the highest antioxidant activity compared to other hydrolysates. To identify antioxidant peptide, peptic hydrolysate was purified using consecutive chromatographic methods, and the antioxidant peptide was identified to be VKAGFAWTANQQLS (1519 Da) by Q-TOF ESI mass spectroscopy. The antioxidant activities of antioxidant peptide from tuna backbone protein (APTBP) was evaluated, and the results show that APTBP significantly inhibited lipid peroxidation in linoleic acid emulsion system and also quenched free radicals (DPPH, hydroxyl and superoxide) in a dose-dependent manner. Moreover, APTBP did not show any cytotoxic effect against MRC-5 and ECV304 cell lines. (C) 2007 Elsevier Ltd. All rights reserved.