Objective: Confocal Raman microspectroscopy (CRM) enables non-invasive depth-scanning of biological tissues. The technique has been used to obtain information about the molecular composition of the skin, tracking of externally applied compounds and to determine molecular concentration profiles. The objective of this study is to use CRM in order to evaluate the changes in stratum corneum hydration when applying polyethylene glycol and the humectants urea and glycerol, and thereby also varying the external chemical potential of water. In the present study we also utilize the advantages of CRM to create novel spatial high- resolution Raman images of stratum corneum. Methodology: Excised porcine skin membranes (500 nm in thickness) were equilibrated from the surface with phosphate buffered saline (PBS) together with different types of humectants using Franz cells. The Raman measurements were performed with a WITec alpha300 system (Ulm, Germany) equipped with a 532 nm laser. The change in stratum corneum hydration after treatment with different humectants was determined from the relative intensity of the water and protein spectra. Raman images were created along a cross section by integrating the Raman intensities for specific vibrational modes. Results and conclusions: The results show that the hydration profiles of stratum corneum correlates well with the gradient in water chemical potential created by the applied humectants, i.e. low molecular weight humectants enable increased hydration of stratum corneum compared to a high molecular weight, non-penetrating polyethylene glycol. In addition the novel results from the Raman imaging experiments illustrates that it is possible to distinguish between water rich domains and the extracellular lipid rich domains along a cross section of the intact skin membrane.