Fusidic acid (FA) is a well-known pharmaceutical antibiotic used to treat dermal infections. This experiment aimed for developing a standardized HPLC protocol to determine the accurate concentration of fusidic acid in both non-ionic and cationic nano-emulsion based gels. For this purpose, a simple, precise, accurate approach was developed. A column with reversed-phase C18 (250 mm x 4.6 mm ID x 5 m) was utilized for the separation process. The main constituents of the HPLC mobile phase were composed of water: acetonitrile (1: 4); adjusted at pH 3.3. The flow rate was 1.0 mL/minute. The optimized wavelength was selected at 235 nm. This approach achieved strong linearity for alcoholic solutions of FA when loaded at a serial concentration ranging from 12.5 to 400 µg/ml. Furthermore, the approach showed good stability and achieved full recovery and an effective separation for FA from the abovementioned formulation. Besides, the protocol validation revealed good robustness at a temperature range of 23 to 27, pH 3.0 to 3.5, detection wavelength 230 to 240 nm, flow rate 0.8 and 1.2 and mobile phase contents of (78:22 to 82:18 acetonitrile/ water). The limit of Detection was obtained 1.33 µg/ml and limit of Quantification was 4.04 µg/ml for FA that uploaded through mentioned formulations. All the validation parameters were within the acceptance criteria, as per ICH , US Pharmacopeia requirements. Overall, an affordable and reproducible method could be achieved for the detection and quantification of fusidic acid within the nano-emulsion based gels formulas.