Here, we describe the isolation of epidermal melanocytes and hair follicle melanocytes from human skin tissue. Epidermal and hair follicle melanocytes are two distinct populations of melanocytes that are contained within the skin and the hair follicle, with differing yet overlapping roles. Epidermal melanocytes are normally isolated from the epidermis of vellus-haired skin tissue, for example, face, breast, abdomen, while hair follicle melanocytes are derived from the outer root sheath (ORS) of the middle/lower terminal anagen hair follicles of the scalp. These methods utilize ethically sourced human skin tissue obtained from donors undergoing plastic surgery.