Oxidative stress is one of the most causes of some severe diseases and the spoilage of foods. Protein hydrolysates containing antioxidant peptides are a suitable candidate to replace synthetic antioxidant. In the present study, the in vitro and cellular antioxidant properties of Saccharomyces cerevisiae protein hydrolysate and the peptide fractions have been reported. The peptide protective role was evaluated in H₂O₂-stimulated Caco-2 cells to consider cell viability, cellular lipid and protein oxidation, and the level of cellular antioxidant enzymes such as Catalase (CAT) and Glutathione-S-transferase (GST). The peptide fractions showed a significant ferric reducing antioxidant power (67.10–93.52 µm FeSO₄/mg protein), and < 3 kDa peptide fraction with 59.5% inhibitory effect on the 7th day, exhibited the most inhibitory activity toward linoleic acid peroxidation. Ultrafiltered fractions ( < 3 kDa and 3–5 kDa) significantly (P ≤ 0.05) decreased the levels of malondialdehyde (MDA) and protein carbonyl and the production of CAT and GST enzymes as protective responses of cells under oxidative stress by H₂O₂. This study confirms the antioxidant activity of yeast protein hydrolysate and peptide fractions and their potential to reduce cellular oxidative stress and thus validates their potential use as a valuable ingredient of functional foods.