Defatted peanut meal hydrolysate (DPMH) was purified using ultrafiltration, gel filtration chromatography, and high-performance liquid chromatography. A tripeptide with strong oxygen radical absorbance capacity (ORAC) was isolated and identified as Tyr-Gly-Ser by ESI-MS/MS. It was then synthesized to measure its antioxidant properties in different systems. The ORAC value of Tyr-Gly-Ser was 3-fold higher than that of glutathione (GSH), and it displayed a stronger protective effect on linoleic acid peroxidation and H(2)O(2)-induced oxidative injury in rat pheochromocytoma line PC12 cells than GSH (p < 0.05). However, Tyr-Gly-Ser showed negligible DPPH radical scavenging activity, reducing power, and no metal chelating ability. The results suggested that Tyr-Gly-Ser displayed antioxidant activity via the hydrogen atom transfer mechanism, and the Tyr at the N-terminal was the hydrogen donor. The ORAC assay was recommended as a reliable and effective method to measure the antioxidant activity in the course of antioxidant peptide isolation.