Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent separation from the remaining primary human keratinocytes culture. Those include rapid adherence of freshly isolated human keratinocytes to collagen type IV through the β1-integrin ligand and Rho-Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49f(bri)/CD71(dim) expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49f(bri)/CD71(dim) subpopulations. The step-by-step methodology herein described indulges the boosting and consecutive purification and separation of interfollicular epidermal stem cells from human keratinocytes cultures.