Citrullus lanatus seed is a potential source of edible protein, hence effective extraction of protein components plays a vital role in food application. Therefore, this study aims for optimization and hydrolysis of protein using single factor and Box Behnken design (BBD). Various concentrations of the protein hydrolysates were studied for antioxidant activity using different assays. The one factor test revealed that important parameters such as alkali concentration (0.8%), temperature (40 °C), time (30 min) and solid to alkali ratio (1:30) enhance the protein concentration from C. lanatus (903.1 mg/g). The maximum degree of hydrolysis 39.30% was achieved at pH 2.4, an enzyme to substrate ratio 3% (w/w), and hydrolysis time 180 min by using BBD design. C. lanatus protein hydrolysate showed 15.71 and 61.67% of DPPH and ABTS radical scavenging activity at 5 mg/ml. It exhibited reducing power optical density 0.18593 nm and metal chelating activity of 42.69% at the concentration of 25 mg/ml. This implied that C. lanatus could be used as a natural antioxidant agent.