Dermal-epidermal separation is an important basic investigation technique for pharmacology, toxicology and biology. To choose the optimal method for a given research need, we reviewed studies on epidermal separation. PubMed, Embase, and Web of Science were utilized. Different separation systems have been compared, including chemical, enzyme, heat and mechanical separation; each has advantages and disadvantages. Heat is simple but causes thermal damage. Chemical reagents are effective but disturb cellular electrolyte equilibrium. Enzymes provide complete separation, but destroy important components. Mechanical division does not alter physical and/or chemical integrity, but necessitates a relatively large sample and may cause cross-contamination of layers. Thus, the appropriate method should be chosen for a given research question, and no single method appears superior for all purposes.