In recent years there has been increased the interest in evaluate antioxidative bioactive peptides derived from food sources, bovine plasma have been shown to possess high antioxidant activity. Oxygen radical antioxidant capacity (ORAC) and trolox equivalent antioxidant capacity (TEAC) assays were compared in order to estimate the antioxidant capacity of bovine plasma hydrolysates (BPH) obtained for Alcalase® 2.4 L. The effect of the degree of hydrolysis (DH) was also evaluated for such activity. Additionally, the peptide fractions with the greatest activity were isolated by using Ultrafiltration Membranes, Ion Exchange Chromatography, and Reverse-Phase High Performance Liquid Chromatography (RP-HPLC). An increase of the degree of hydrolysis was found for these methods that correspond to an increase in the antioxidant activity; besides, the high relationship between ORAC and TEAC assays, with a correlation coefficient of 0.974, fitted a double squared model. A peptidic fraction with highest antioxidant capacity was obtained by separation processes, with values 4457.9 ± 72.5 and 1409 ± 43.41 µmolTE/g for ORAC and TEAC methods respectively, and contained peptides with sequences Q(K)Q(K)PVRDQ(K) and GAHQPSG, which were determined by MALDI-TOF-TOF and manual nESI-MS/MS. .