The aim of this work is to validate the chromatographic analytical method for the determination of sulfamethoxazole in water on a HPLC-DAD instrument. Validation of a previously developed method is important because it provides confirmation of the reliability and quality of the method, which can be used with confidence in the future when analyzing a particular sample on a particular instrument. The analytical system in this work was validated by examining the method performance characteristics, i.e., validation parameters such as selectivity, linearity, sensitivity, limit of detection, limit of quantification limits, precision, trueness, working range, and robustness. Based on the results, the correctness of the investigated analytical method is concluded. To determine the above criteria, a quantitative determination of the antibiotic sulfamethoxazole dissolved in deionized and spring water was performed. Each experiment was carried out with samples in both types of water in order to compare the results of experiments with deionized water, which is characterized by high purity, with the results of experiments with spring water, which contains various interferences. Based on the research results, it is concluded that the mentioned method is reliable and correct, or, in other words, it is validated. The research in this work will serve for further tests of analysts who have the task to qualitatively and quantitatively determine the drug sulfamethoxazole in water samples by high-performance liquid chromatography with a diode array detector.