The human hair follicle (HF) harbors distinct populations of epithelial stem and progenitor cells that are essential for hair homeostasis and epidermal wound healing. While the follicular niche, the anatomical equivalent of the murine bulge located at the insertion site of the arrector pili muscle, has historically been the primary target for the isolation of hair follicle stem cells (HFSCs), capturing the full epithelial stem and progenitor hierarchy within the outer root sheath (ORS) is critical for a comprehensive understanding of human hair follicle biology. As integrin α6 (CD49f) is a well-established surface marker associated with basal epithelial stem and progenitor states, and long-term proliferative potential of immature keratinocytes, we have exploited this marker to isolate an epithelial cell population corresponding to the keratinocyte basal layer of the ORS. This chapter describes a protocol for the enrichment of stem and progenitor cells from intact human hair follicles, combining manual microdissection of the entire hair follicle, enzymatic dissociation, and flow cytometry sorting based on CD49f expression. Functional validation using in vitro proliferation and clone-forming assays showed that the CD49fhigh fraction exhibits significantly greater proliferative and clonogenic capacity, compared to the ORS total and CD49flow populations. This method preserved cell viability and phenotypic integrity. Consequently, the isolated keratinocyte populations are suitable for a broad range of downstream uses and applications, including transcriptomic analyses such as single-cell RNA sequencing (scRNA-seq), functional assays, and regenerative studies. Altogether, this workflow provides a robust platform for future approaches dedicated to the treatment of alopecia, promotion of hair longevity, and for tissue engineering applications.