Epidermal stem cells are of major importance for tissue homeostasis, wound repair, tumor initiation, and gene therapy. Here we describe an in vivo regeneration assay to test for the ability of keratinocyte progenitors to maintain an epidermis over the long-term in vivo. Limiting dilution analysis of epidermal repopulating units in this in vivo regeneration assay at sequential time points allows the frequency of short-term (transit amplifying cell) and long-term (stem cell) repopulating cells to be quantified.