Amino acids are the fundamental units that proteins are composed of. They are organic molecules essential for life and play vital roles in our bodies. Some amino acids are produced by our bodies (non-essential amino acids), while others need to be obtained through food (essential amino acids). In addition, these molecules perform crucial functions, such as speeding up biochemical reactions (enzymes), acting as chemical signals (hormones), forming structural components (muscles, tendons, and nails), and conferring diversity to each specific protein [1]. Methods for the derivatization and analysis of amino acids often involve chemical modification to improve detection sensitivity, with techniques such as high-performance liquid chromatography (HPLC) being typical. However, the derivatization process can be problematic for specific amino acids, leading to incomplete reactions or the instability of the derivatives. This work aims to optimize the amino acid derivatization reaction in food matrices through high-performance liquid chromatography (HPLC) analysis to improve the sensitivity and precision of amino acid quantification. To optimize the amino acid derivatization reaction, a systematic approach was used, varying the concentrations of OPA and 3-MPA (10, 15, 20, 25, and 30 mM), as well as the concentration of FMOC-Cl (1, 2, 5, and 10 mM) and the reaction time (0.5, 1, 1.5 and 2 min). In the HPLC system, an acetate buffer was used as the mobile phase, and a solution of acetonitrile, methanol, and water was used. With this experimental method, it was possible to identify the ideal conditions using a specific combination of concentrations of OPA, 3-MPA, and FMOC-Cl, together with the time of occurrence, which was investigated for greater sensitivity and precision in the quantification of amino acids. This study improved the methodology for analyzing amino acids in food matrices using HPLC, enabling more reliable and accurate results.