Stem cells antigen-1 (Sca-1) is an 18-kDa mouse glycosyl phosphatidylinositol-anchored cell surface protein that has widely been used for isolation of murine hematopoietic stem cells (HSCs). However, recently several studies have reported the expression of this protein in non-HSCs such as the liver, skin and muscles among others. In the current study, we used fluorescence-activated cell sorting (FACS) technique to sort Sca-1+ stem cells from mouse hindlimb muscles. The Sca-1+ cells were divided into two sets first set of Sca-1+ cells were processed for protein extraction immediately after sorting (ex vivo set) while the second set of samples, referred to as in vitro set, were expanded in cell culture after sorting and harvested after two passage and then processed for protein extraction. A high-resolution proteome map of the ex vivo and in vitro sets was generated Orbitrap Fusion Tribrid Mass Spectrometer. In total, the analysis led to the identification of 5,581 protein groups.