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An isocratic RP-HPLC–UV method for the analysis of voriconazole in skin diffusate samples has been developed and validated. Experimental design was employed to optimize the method. The method was validated as per ICH guidelines. Linearity was observed over the concentration range of 2–100 μg mL⁻¹ (r² = 0.999). Limits of detection and quantification were 0.6 and 2 μg mL⁻¹, respectively. Intra-day and inter-day precision (% RSD) was within the ICH limits (⩽2%). The method was successfully used to analyze skin diffusate samples, and the effectiveness of different permeation enhancers was compared with respect to flux and permeability coefficient.