Satellite cells are a heterogeneous population of muscle progenitors with stem cell properties responsible for the regeneration of adult skeletal muscle. Increasing interest in the therapeutic potential of satellite cells has challenged researchers with the need to purify a homogenous population of muscle progenitors. Here we provide a detailed protocol for the isolation of a pure population of satellite cells using fluorescence activated cell sorting. We give specific guidelines to ameliorate the reproducibility of the satellite cell isolation protocol with the goal to standardize procedures across labs. This protocol identifies satellite cells within adult skeletal muscle as an enriched population of Integrin α7+ /CD34+ double positive cells and CD45, CD31, CD11b, and Sca1 negative (Lin− ) cells (Integrin α7+ /CD34+ /Lin− ).. Functional assay shows that Integrin α7+ /CD34+ /Lin− satellite cells possess high myogenic potential and ability to regenerate muscle depleted satellite cells upon transplantation.