ABSTRACT The objective of this research was to evaluate the effect of increasing levels of associated glutamine and glutamic acid on growth performance and intestinal development of Nile tilapia, Oreochromis niloticus, fingerlings. Five isoproteic (~344.70 g kg−1 crude protein) and isocaloric diets (~3,925 kcal kg−1 gross energy) were developed containing 0, 5, 10, 15, or 20 g kg−1 of associated glutamine and glutamic acid in extruded diets. Fish (n = 2,000, mean body weight of 2.12±0.53 g) were distributed into twenty 1-m3 floating net cages in an entirely randomized design with five treatments and four replicates, and each replicate comprised one floating net cage with 100 fish. Fish were hand-fed seven days per week, three times a day until apparent satiety for 45 days. There was a quadratic effect on final body weight, body weight gain, daily weight gain, feed conversion ratio, protein retention efficiency, net protein utilization, and intestinal villi height with optimized values for supplementation of associated glutamine and glutamic acid at 10.77, 10.67, 10.00, 8.85, 9.85, 10.15, and 10.98 g kg−1, respectively. There was no effect of associated glutamine and glutamic acid supplementation on feed intake, survival, and body composition. We conclude that 10.67 g kg−1 of associated glutamine and glutamic acid is adequate for growth performance optimization, and supplementation at 10.98 g kg−1 exerts trophic action and improves intestinal morphometry in cage-farmed Nile tilapia fingerlings.