Autologous cell therapy is a promising approach for accelerating the regeneration of chronic and acute skin wounds. The effectiveness of such treatments depends largely on the availability of large numbers of viable, minimally manipulated cells that have retained their natural physiological properties. Fibroblasts and keratinocytes are key cells in the wound healing process, responsible for extracellular matrix deposition and re-epithelialization, respectively. This chapter describes in detail an integrated protocol for the highly efficient enzymatic isolation of these cells from skin biopsies, followed by the application of a new adhesion-based separation method with temporal resolution. The enzymatic protocol uses an optimized cocktail of dispase and collagenases for efficient tissue digestion, yielding up to 8.9–9.1 million viable cells per cm2 with >90% viability. The subsequent method of separating these cells is based on the differential kinetics of keratinocyte and fibroblast adhesion to enrich populations without antibodies or complex equipment, achieving a 30–40% increase in fibroblast purity.