Nuclear microscopy is a technique based on a focused beam of accelerated particles that has the ability of imaging the morphology of the tissue in vivo and of producing the correspondent elemental maps, whether in major, minor, or trace concentrations. These characteristics constitute a strong advantage in studying the morphology of human skin, its elemental distributions and the permeation mechanisms of chemical compounds. In this study, nuclear microscopy techniques such as scanning transmission ion microscopy and particle induced X-ray emission were applied simultaneously, to cryopreserved human skin samples with the purpose of obtaining high-resolution images of cells and tissue morphology. In addition, quantitative elemental profiling and mapping of phosphorus, calcium, chlorine, and potassium in skin cross-sections were obtained. This procedure accurately distinguishes the epidermal strata and dermis by overlapping in real time the elemental information with density images obtained from the transmitted beam. A validation procedure for elemental distributions in human skin based on differential density of epidermal strata and dermis was established. As demonstrated, this procedure can be used in future studies as a tool for the in vivo examination of trans-epidermal and -dermal delivery of products.