The phenolic content and antioxidant activity of grape (Vitis vinifera) seed powder extracted by in vitro physiological procedure and chemical procedure were investigated. The antioxidant potential of the extract were assessed by employing different in vitro assays such as CUPRAC, DPPH, ABTS, OH radical scavenging capacity, and peroxidation inhibiting activity. The in vitro physiological procedure yielded a higher phenolic content and antioxidant capacity than the chemical procedure. As for digestive enzymatic extracts, phenolic content and antioxidant capacity of the dialysates of grape seed powder were lower than those of the retentates. As for solvents extracts, extraction with acetone:water (70:30) led to the maximum phenolic content and antioxidant capacity, while water gave the lowest phenolic content and antioxidant capacity. Our results suggest that the biological properties of natural antioxidants determined by in vitro physiological procedure may be more useful for nutritional purposes than the values determined in solvent extracts.