Alpha_s2-casein is particularly rich in π electron rich aromatic amino acids that, in the absence of structural constraints, partake in π-π stacking and interact with positively charged residues (cation-π) resulting in electronic continuity required for rapid quenching of lone-pairs of oxidative radicals. This study investigated isolation of α_s2-casein from bovine milk, standardization of its chymotryptic hydrolysis, visualization of resulting peptide profiles by tricine-SDS-Polyacrylamide gel electrophoresis (TSDS-PAGE) and size-exclusion high performance liquid chromatography (SE-HPLC), and determination of the antioxidative efficacy of the fractions. Alpha_s2-casein was isolated from whole casein by a 2-step 1-proponal-precipitation method and hydrolyzed using chymotrypsin (EC 3.4.21.1) because it selectively cleaves peptide bonds adjacent to aromatic residues, increasing the chances for inter-peptide aromatic-stacking. Five mg/mL of α_s2-casein was hydrolyzed with chymotrypsin (50 μg/mL) for 5–60 min at 37°C. A novel fixing method with sodium hypochlorite was used to fix the highly amphipathic peptides after TSDS-PAGE and visualization was by silver staining. The original protein was complete hydrolyzed to smaller peptides within 5 min. Peptide profiles determined by SE-HPLC, using a Superdex Peptide 10/300 GL column and 30% acetonitrile (v/v) as the eluent, substantiated the effectiveness of the peptide fixation following TSDS-PAGE peptide profiling. Average molecular weight of each fraction was determined by comparing its retention time with those of molecular weight standards. Data depicted an inverse relationship between molecular weight and the duration of enzymatic hydrolysis. Average molecular weight of the smallest fraction at 5 min of hydrolysis was 981 Da, and it was gradually reduced to 130 Da after 60 min of hydrolysis. As expected all hydrolyzates had oxygen radical absorbance capacity values (2000–3000 μM TE) that were significantly (P < 0.05) higher than that of the original protein (580 μM TE). Correlation of the antioxidative efficacy with residual secondary structural constraints and intensity of association tendency of the peptide fractions is presently being investigated.