Understanding amino acid (AA) supplementation, including glycine (Gly), is important to optimize fish growth performance as alternative protein sources become more prevalent in the aquaculture industry. A 12-week feeding trial evaluated the effect of dietary Gly supplementation on growth performance and blood biochemistry of juvenile Florida pompano (Trachinotus carolinus; 19.55 ± 0.32 g). Experimental diets (40% crude protein and 8% crude lipid) incorporated soybean meal, poultry by-product meal, and corn protein concentrate. The Gly was supplemented at 0%, 0.25%, 0.5%, and 1% and alanine (Ala) was adjusted to ensure all diets were isonitrogenous. Following the conclusion of the trial, final weight (85.21 – 90.93 g), weight gain (336% – 366%), and feed conversion ratio (1.61 – 1.69) showed no significant differences among treatments. A significant linear decrease in hepatosomatic index (R2 = 0.244, p = 0.027), relatively stabilized liver enzymes, and a significant increase in serum cholesterol at 1% Gly supplementation (R2 = 0.507; p = 0.002) suggest that Gly may influence hepatic metabolism. This potentiation may be through enhanced bile acid conjugation, revealing additional research questions. Whole-body proximate composition and AA profiles remained unchanged, and serum lysozyme activity showed no significant variation across treatments (p = 0.730). These findings suggest that Gly supplementation did not enhance growth but influenced some metabolic parameters. Further research with Florida pompano under various culture conditions and stress challenges should help optimize Gly supplementation for this species.