In this study, we interrogated the protein composition of exosomes purified from cell culture supernatants of 5 different RMS cell lines, by LC-MS/MS technology and analysis using bio-informatics tools. We attempted to characterize the differences and similarities between exosomes derived from ERMS and ARMS cells, and common as well as unique characteristics of RMS exosome protein content when compared to that of other cancer and non-cancer cells. Our results identify potential protein exosome biomarkers for RMS to be further investigated in future clinical studies.