The effects of ultrasound (US) (40 kHz, 23.8 W/L) on commercial protease structures and their impact on pumpkin seed protein (PSP) hydrolysis were evaluated, as well as the solubility and antioxidant activity of hydrolysates. The ultrasonic pre-treatment activated Neutrase® (22.7 %), Brauzyn® (20.8 %) and Flavourzyme® (22.4 %), with more significant changes in the tertiary structure of Flavourzyme®, which also showed the highest increases in particle size (∼21 %) and polydispersity index (PDI) (∼60 %) compared to its native form. On the other hand, US pre-treatment under inactivation conditions resulted in substantial structural changes, indicating protein denaturation with the formation of heterogeneous and strongly electronegative aggregates. Proteolysis of PSP with US-activated enzymes showed a higher degree of hydrolysis (DH) (up to 75 %) and the obtained peptides showed higher solubility (up to 53 %) and antioxidant activity (up to 40 %). The findings emphasize that applying ultrasound under specific conditions can induce moderate structural changes in proteases, enhancing their hydrolytic efficiency.