Study of stem cell phenotype and functions requires their proper isolation. Stem cells isolated from skeletal muscle are a useful tool to explore molecular pathways involved in the regulation of myogenesis. Among progenitor cells, a subset of cells, called reserve cells, has been identified, in vitro, in myogenic cell cultures. This subset of cells remains undifferentiated while the main population of progenitor cells commits to terminal myogenic differentiation. When replated, these reserve cells grow as new colonies of progenitors. At the time of differentiation, they reform both differentiated myotubes and undifferentiated reserve cells. Here, we present a protocol to obtain and further isolate reserve cells from both human and murine myogenic cell cultures, together with techniques to analyze their cell cycle status.