The physicochemical properties of the stratum corneum (SC) assures skin barrier integrity and can be affected by surfactants-containing cleansers by modifying skin pH and components.The aim of this study was to assess the effect of cleansers on the surface of human skin by confocal laser scanning microscopy (CLSM) in the fluorescence mode, by monitoring the labelling of acridine orange dye through the SC. First, we evaluated the application of SDS solutions (0.1-0.5-1-5%). We then compared 0.5% SDS solution with a body shower cleanser (product A) or an emollient shower oil containing Rhealba® Oat Plantlet extract for soothing properties (product B). SDS or test products diluted with acridine orange dye were applied on the surface of skin biopsies. After washes, CLSM imaging was proceeded with the Vivascope®2500 (MAVIG). Five VivaStack acquisitions were performed per condition. For quantification of dye penetration, the stack projections were analyzed using ImageJ and internal Octopus softwares. The experiments were repeated on n=3 donors. Untreated skin biopsies exhibited a low level of dye penetration and this was expected for a normal, unmodified skin barrier. In contrast, we observed a significant, dose-dependent- effect, increase of dye penetration through SC exposed to SDS (x4.1, x24, x48, and x46), revealing alterations of the skin barrier. Comparison of test products showed that 0.5% SDS and product A induced a significant increase of dye penetration (x11.3 and x6, respectively) while no significant modification was found for product B, demonstrating that emollient shower oil containing Rhealba® Oat Plantlet extract preserved SC and skin barrier. Our data showed that fluorescent dye penetration monitored with Vivascope®2500 CLSM imaging is a promising method to evaluate the effect of cosmetic products on skin barrier.