A 4 x 3 factorial study was conducted to evaluate the effect of four experimental diets (a control diet and a 0, 50, and 100% fish meal replacement diet) and the period of time Nile tilapia, Oreochromis niloticus, were fed those diets (0, 20, and 40 days) on the alkaline proteolytic activity of the animals' digestive tract, as well as their potential interaction. Significant differences (at P < 0.05) and a significant interaction were observed among dietary treatments for the alkaline proteolytic activity of tilapia after 40 days of feeding. This study confirmed that, under these experimental conditions, a 50% fish meal replacement formulation elicited the highest alkaline proteolytic activity in the digestive tract of tilapia, which resulted in the highest final weight and specific growth rate (SGR), but further research is needed to establish the relative contribution of the alkaline proteases to the overall proteolytic activity of this omnivorous fish species.