The human skin is a complex organ composed of multiple layers and specialized cell types. At its core, the skin’s function is maintained by the interplay between two key compartments: the epidermis, that is composed of suprabasal and basal keratinocytes, and the dermis, which is inhabited by a heterogeneous population of fibroblasts. Additionally, the skin contains stem cell-bearing hair follicles that span across both the epidermis and dermis, providing a vital source of cellular renewal. Each of these niches—the epidermis, dermis, and hair follicles—individually and collectively contribute to skin integrity, homeostasis, and regeneration. However, dermal fibroblasts can also undergo malignant transformation in pathological conditions such as skin cancer development, giving rise to cancer-associated fibroblasts (CAFs) that promote tumor progression and may mediate therapeutic resistance. To study key aspects of skin cell populations/niches, in vitro culture systems offer a controlled, convenient platform. We here summarize standardized protocols for the isolation of primary human epidermal keratinocytes, dermal fibroblasts, and hair follicles from healthy skin, as well as CAFs from cancerous human skin. We describe methods for feeder-free keratinocyte culture as well as the culture of dermal fibroblasts and CAFs. In addition, we provide a detailed description of functional assays, including three-dimensional (3D) culture, flow cytometry, and migration analysis of primarily isolated skin cells and immunofluorescent staining of individually isolated hair follicles. These protocols provide a versatile toolkit to investigate skin biology wound healing, epithelial-mesenchymal interactions, and tumor microenvironment dynamics under physiologically relevant conditions.