The dual-energy X-ray absorptiometry (DXA) is an alternative to the comparative slaughter technique (CST), allowing the analysis of body chemical composition of fish in vivo. The present work aimed to standardize the dual-energy X-ray absorptiometry (DXA) technique in fish and determine the body chemical composition of Pacu (Piaractus mesopotamicus) and Nile tilapia (Oreochromis niloticus) during different growth stages in vivo. By the relationship between chemical compositions of fish determined by CST and DXA linear equations were generated, where Y-axis represented the results of CST and X-axis represented the results of DXA. This relationship was determined for each chemical component including water, protein, mineral matter, and fat. The deviation composition (D, g) obtained from the analysis of the multiple linear equations, and the correlation of prediction (R2) for Nile tilapia and Pacu were, total body weight of tilapia: D = 1.12 g, R2 = 0.99; Pacu: D = 1.53 g, R2 = 0.98; protein of tilapia: D = 1.87 g, R2 = 0.96; Pacu: D = 0.61 g, R2 = 0.99; water of tilapia: D = 2.09 g, R2 = 0.95; Pacu: D = 1.35 g, R2 = 0.99; fat of tilapia: D = 1.37 g, R2 = 0.99; Pacu: D = 0.67 g, R2 = 0.99, and mineral matter of tilapia 0.71 g, R2 = 0.98; Pacu: D = 0.37 g, R2 = 0.98. These values show high correlations (R2 > 0.9) and precision in the evaluation of the body components and weight variables. This study provides important information about the substitution of the CST with the DXA which will help to determine the body composition of fish in vivo in a more precise way, with less operational costs and in a short time.