Enzymes may offer great potentials in topical pharmaceutical applications provided that treatment conditions are controlled for efficacy and safety. In this study, the effect of alkaline protease produced by recombinant Bacillus subtilis cells on the ex-vivo permeability of rabbit ear skin was investigated under different conditions of enzyme activity (5–60 units) and exposure time (15–60 min). Data for transepidermal water loss (TEWL) and permeation of a hydrophilic dye, rhodamine B (Rb), indicated biphasic activity-dependent and exposure time-dependent skin permeability. Maximum effects were obtained at 20 proteolytic units and 30 min exposure. Findings proved consistent with histopathological changes indicating progressive stratum corneum (SC) loss and disruption of the dermo-epidermal junction at 20 units and up to 30 min exposure time followed by dermal hyalinization at longer exposure. This was associated with progressive loss of skin hair. Applying the identified pretreatment conditions to transdermal delivery of vardenafil in a gel base across dorsal rat skin indicated a significant increase in plasma levels at 30 and 60 min with minimal histopathological changes 5 days post enzyme treatment. Accordingly, the recombinant B. subtilis alkaline protease offers promise as a pharmaceutical enzyme for transdermal drug delivery bioenhancement and dermatological applications.