In present study, whey protein hydrolysate was prepared using Alcalase 2.4L from whey protein concentrate. The effect of temperature (40, 45, 50 and 55oC), time (30, 60, 90, 120, 150, 180 and 210 m) and enzyme/substrate ratio (30, 60 and 90 Anson unit/kg protein), on degree of hydrolysis and antioxidant activity of product were investigatedin a completely randomized design. The highest degree of hydrolysis was observed at 55oC, hydrolysis time of 180 minutes andenzyme/substrate ratio of 60 Anson unit/kg substrate. Under these conditions, degree of hydrolysis was 51.62%. The antioxidant activity of protein hydrolysate was studied using reducing power and Fe2+chelating activity. At maximum degree of hydrolysis, Fe2+ chelating activitywere obtained 50.41%. As well as under this condition reducing-power of protein hydrolysate was 0.156 which showed 20.47% reducing-power compared to 100 ppm ascorbic acid.