ABSTRACT Autolysis-assisted process mediated by indigenous protease in combination withthermal hydrolysis and hydrolysis using papain was used for production of gelatinhydrolysate from skin of unicorn leatherjacket. Prior autolysis could enhance theyield and antioxidative activity of gelatin hydrolysates, especially when subsequenthydrolysis by 2% papain was implemented (AU + HP2.0). Hydrolysates possessedABTS (2,2′-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid]) radical and H 2 Oscavenging activities as well as metal chelating activity. AU + HP2.0 and gelatinhydrolysate prepared using papain without prior autolysis (HP2.0) could retardoxidation in lecithin liposome system, and antioxidative activity of hydrolysatesat 10.0 g/L was comparable to that of 0.1 g/L Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), in which the oxidation was almost com-pletely inhibited. On the basis of size exclusion chromatography, antioxidativepeptideinAU+ HP2.0 had molecular weight of 2200 Da. Therefore, autolysis-assisted process showed the potential for the production of gelatin hydrolysateswith enhanced antioxidative activity.