The aim of this research is to determine the optimum concentration of the coupling agent used in chloramphenicol analysis, then calculate the limit of detection of that photometric reaction. Chloramphenicol is a colorless compound that is often abused in animals whose products are consumed by humans. Chloramphenicol has side effects on the human body. So, chloramphenicol must be analyzed. To facilitate the analysis process, chloramphenicol must be converted into colored compounds called azo compounds. To make azo compounds must go through the process of reduction, diazotization, and coupling. A coupling agent is an important compound that played a role in an azo compound formation and was analyzed using UV-Vis spectrophotometer. This research, used N-(-1-Naphthyl) ethylenediamine dihydrochloride (NEDA) with variations concentrations of 0.1%, 0.3%, 0.5% at 565 nm. The optimum coupling concentration that gives maximum absorbance was 0.1%. The detection limit (LOD) obtained by using the optimum coupling is 0.0498 μg/mL, the limit of quantitation (LOQ) is 0.1660 μg/mL, the sensitivity value is 0.1425, and a correlation coefficient (R2) of 0.999 with a concentration range of chloramphenicol is 0,14 - 1,4 ppm.