Human skin consists of three compartments, each endowed with a particular structure and the presence of several immune and nonimmune cells that together comprise a protective shield and orchestrate multiple processes in the skin. Appropriate processing of human skin samples acquired from healthy volunteers or patients is essential for successful analysis in basic, translational, and clinical research to obtain accurate and reliable results, despite differences between individuals. From the wide

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... range of available assays and methods, it is necessary to select the suitable method for separation of skin compartments, which will provide preservation or high viability of skin cells or whole structures that will be analyzed or further processed. In this paper, we review and discuss skin separation methods and compare their features such as processing time, cell viability, location of the basement membrane after detachment of the epidermis from the dermis, and their application. Furthermore, we visualize different cell populations and structures in epidermal and dermal sheets using confocal microscopy. It is aimed to provide an overview of the optimal processing of human skin samples and their possible application.