Gramicidin is a polypeptide antibiotic composed of a mixture of antimicrobial compounds. Thus, its antibacterial activity is preferentially assessed using a microbiological assay. The aim of this study is targeting to establish and validate a microbiological potency for Gramicidin with a view to the employment of a simple method with more than two folds output per test run (if compared with symmetrical designs) using 3×1 experimental designs with reasonably statistically acceptable results. The validation criteria of gramicidin turbidimetric assay using the USP method were tested in terms of selectivity, linearity, accuracy, precision and robustness. Moreover, the consistency of the experimental groups was examined in terms of error and difference from the target labelled concentration of 0.25 mg g-1 value, in addition to the uncertainty factor. Verification of the assay suitability was evaluated statistically against reference antibiotics of known activity. Calibration of the analytical curve showed a coefficient of correlation (r) = 0.9980 with none of the relative standard deviations (RSD) values greater than three. There was no observable fixed or variable deviation in the absorbance measurement with concentration increment. The accuracy output and profile were evaluated over ranges 50%, 100% and 150% having a maximum RSD of around three with reasonable results, confidence and absence of concentration-related bias. Robustness, precision and suitability verification were evaluated with no outliers and all RSDs below five. The turbidimetric assay design of 3×1 for gramicidin showed acceptable validation parameters and could be used as a substitute design for conventional higher-level parallel line assay models.