The use of skin as an alternative route for administering systemically active drugs has attracted considerable interest in recent years. However, the skin provides an excellent barrier, which limits the number of drug molecules suitable for transdermal delivery. Thus, in order to improve cutaneous delivery, it is necessary to adopt an enhancement method, either (i) passively using novel formulations, e.g. microemulsions, liposomes, and colloidal polymeric suspensions, or more conventional skin permeation enhancers, or (ii) with a physical approach, such as, iontophoresis, sonophoresis or electroporation. Although there has been much progress, the precise modes of action of the different techniques used are far from well-understood. The objective of this review, therefore, is to evaluate how confocal laser scanning microscopy may contribute to the determination of the mechanisms of diverse skin penetration enhancement strategies.