A significant stride in enhancing the care for extensively burned patients has been made through the cultivation of autologous keratinocytes, resulting in the formation of cohesive epithelial tissue sheets. Beyond clinical applications, both keratinocyte and melanocyte cultures play vital roles in investigating diverse cellular and molecular mechanisms underlying various skin pathologies. In epithelial cell culture, preserving stem cells is crucial, necessitating dependable methods and optimal conditions. Well-cultured keratinocytes typically exhibit consistent cuboid morphology and maintain proliferative capacity over numerous passages. In contrast, melanocyte culture requires specialized additives to stimulate growth, sustain differentiation, and ensure purity. This chapter provides a comprehensive guide to the materials and techniques essential for efficient keratinocyte culture for clinical purposes and melanocyte culture for research endeavors. Covered aspects include tissue sampling and transportation, isolation, routine culture, subculture, and cryopreservation.