As natural bioactive compounds with recognized antioxidant power, polyphenols may impact on several diseases associated with oxidative stress. In this study, defatted sesame flour (DSF) was investigated as a potential source of dietary polyphenols and a functional ingredient. Additionally, the impact of manufacture and digestion processes on the biological efficacy of the resulting extracts in the HepG2 cell line was explored. For this purpose, polyphenolic extracts were obtained from three sources: DSF, a control sweet cookie formulation and 10% DSF-enriched cookies. In addition, extracts of potentially bioaccessible polyphenols from in vitro digested cookies were collected. Cells were incubated with all these extracts and then injured with H2O2. Intracellular oxidative state and viability, antioxidant enzymes activities, glutathione (GSH) content, and oxidation of proteins and lipids were analyzed. The results showed DSF pro-oxidant actions on the cellular redox state, which hormetically enhanced the antioxidant response. Similarly, biological activity of DSF enriched cookies was found to persist after manufacture and digestion, promoting lipid peroxidation alleviation and GSH replenishment.By promoting an antioxidant response in cells, DSF may be considered for functional incorporation, offering preventive benefits and protective action to counterbalance oxidative damage. This study contributes to highlighting the benefits of DFS validating its final biological effects in a cellular model.