BACKGROUND Mucuna pruriens L. is a legume sown in the Mexican southeast with an important protein content. Studies have shown the potential use of by‐products derived from Mucuna as a functional food because of the hypoglycemic and antihypertensive activities. Thus, this study aims to assess the antioxidant and protective effect of the peptide fractions derived from M. pruriens L., in vitro on the HeLa cell line. An enzymatic hydrolysis with pepsin–pancreatin was performed on the total protein concentrate, from which five peptide fractions were obtained. RESULTS All protein derivatives from M. pruriens L., except F5–10 kDa, decreased the hydrogen peroxide production by more than 50%. The highest antioxidant activity was exhibited by F1–3 kDa, which lowered the intracellular reactive oxygen species by 207 ± 4.20%. No significant differences were found in the protective effects of the protein hydrolysate, F5–10 kDa, F3–5 kDa and F1–3 kDa relative to the N‐acetylcysteine control group. CONCLUSION This elucidated the potential action mechanisms of M. pruriens L. protein derivatives for future investigations and their role in the prevention and treatment of oxidative stress. © 2019 Society of Chemical Industry