N-acetylmuramoyl-L-alanine amidase (NAM-amidase) is integral to bacterial cell wall metabolism and represents a promising target for novel antibacterial strategies. However, the reliable quantification of its activity is often hindered by the limitations of conventional spectrophotometric assays, such as limited specificity and matrix interferences. Here, we report on the development, optimization, and validation of a cost-effective high-performance liquid chromatography (HPLC) method for measuring NAM-amidase activity by detecting its enzymatic product, p-nitroaniline. A Design of Experiments (DoE) approach-specifically, Plackett-Burman followed by Box-Behnken designs-enabled the systematic identification and optimization of critical method variables. The resulting protocol employs an isocratic mobile phase of methanol and o-phosphoric acid in a 10 cm RP-18 column, requiring only standard UV-vis detection and yielding a short analysis time (8 min). Method validation according to ICH Q2(R2) guidelines showed excellent linearity (R2 = 0.9999) from 0.1 to 100 μM p-nitroaniline, corresponding to 0.04-40.0 U per mL NAM-amidase. The method exhibited a 0.033 μM limit of detection, with strong accuracy, precision (RSD < 2%), and robustness. Additionally, precision, stability, and method robustness were evaluated on real samples of an in-house isolated and purified amidase preparations, with all acceptance criteria being met. These results demonstrated the suitability of the developed method for practical laboratory application. Notably, the method's minimal reagent requirements and moderate instrumentation needs broaden its suitability for a variety of laboratory settings, including those with limited resources. This, combined with high specificity, wide linear range, and stable performance, underscores its potential for both routine and advanced applications, including enzymatic assays in basic research, diagnostic evaluations, and pharmaceutical development.