Some bioactive peptides have been identified by activity-guided fractionation. However, it is difficult to prepare or synthesize enough amounts of active peptides for food ingredients and for animal or human trials. The objective of the present study was to prepare peptide fractions of sufficient amounts with high antioxidant activity which could be applied to the food and animal model. Oyster hydrolysate was fractionated the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing (autofocusing). To monitor the fractionation of peptides by autofocusing, amino acid analysis of the autofocusing fractions was performed. Each autofocusing fraction was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl (OH) radical scavenging activities, oxygen radical absorbance capacity (ORAC), and Fe 2+ chelatingassays. More than 40% of the peptides were distributed between Fr. 4 and 7 (pH<5.0). Approximately 16% of the peptides were recovered in basic fractions (pH>10.0; Fr. 9-10). 11 % of peptides were recovered in the neutral fraction (6.0<pH<7.0; Fr. 8). The peptides in the acidic and basic fractions are characterized by a higher content of acidic and basic amino acids, respectively. Acidic fraction showed higher ORAC values than crude oyster hydrolysate. Also, the acidic fraction showed high DPPH and OH radical scavenging activity. On the other hand, basic fraction showed higher chelating ability than crude oyster hydrolysate. In the present study, oyster peptide fractions of large-scale with high antioxidant activity were successfully fractionated by autofocusing for food additives and animal trials.