The microbiological assay is one of the most commonly used techniques for potency determination using biological means through indicator microorganisms in research, clinical, industrial and quality control activities. The current work aimed to validate the determination of Neomycin Sulfate potency through the agar diffusion method using the 2 x 2 Parallel Line Model. The study investigates both the quality of results as a zone of inhibition and the inspected validation parameters which consist of specificity, linearity, accuracy, precision (in terms of repeatability and intermediate precision) and robustness. The initial step determined the validity of raw data for further analysis in terms of normality using QQ plot, homoscedasticity by Cochran's test and the absence of true outlier values through both G statistics (USP), Robust regression and Outlier removal tests. The assay design and procedure showed selectivity towards the intended antibiotic and the calibration curve showed acceptable linearity (r = 0.998) without any fixed or relative concentration-related bias. Accuracy profile covering 50, 100 and 150% of the target concentration demonstrated potencies with a confidence interval that is contained within the acceptance limits. Results of the precision and robustness showed RSD% ˂2.00. The assay design is suitable for the assay of Neomycin Sulfate.