The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein hydrolysates (SPHs). Scorpion protein (SP) was first hydrolyzed by trypsin, papain, and alcalase, respectively. Results from hydrolysis tests revealed that peptides hydrolyzed with papain showed the highest degree of hydrolysis (DH), yield and antioxidant activity. The effect of papain hydrolysis on scorpion protein was optimized using the response surface methodology. The highest DH (31.31%) and yield (52.02%) of SPHs were obtained under the following conditions: hydrolysis time, 4.0 h; hydrolysis temperature, 50 degrees C; and enzyme/substrate ratio, 2.43%. Ultrafiltration, gel filtration and reversed-phase high-performance liquid chromatography was used and two novel antioxidant peptides were obtained. The sequences of the peptides determined by MALDI-TOF-MS/MS were LPTETLH (MW: 810.43 Da, P4-1) and IEEDLER (MW: 903.44 Da, P4-2), respectively. The results revealed SPHs as a potential valuable bioresource for production of antioxidant peptides in the food system.