The plant protease actinidin has been frequently used in the food industry, but its application in dairy systems remains largely unassessed. The aim of this research was to establish the effect of temperature (15–60 °C), time (0–5 h) and enzyme-to-substrate ratio on the actinidin-induced hydrolysis of proteins in whey protein isolate (WPI), whey protein concentrate (WPC) and milk protein concentrate (MPC), as monitored through the degree of hydrolysis (DH) and SDS-PAGE. The DH increased with increasing temperature and incubation time for all three protein sources. A lower E:S ratio resulted in a greater DH for WPC and WPI, but not for MPC. SDS-PAGE analysis revealed that actinidin mainly acted on α-lactalbumin and αS-caseins in WPI and MPC, respectively.