Extracellular vesicles (EVs) hold value as accessible biomarkers for understanding cellular differentiation and related pathologies. Herein, EV biomarkers in models of skeletal muscle dormancy and differentiation have been comparatively profiled using Raman spectroscopy (RS). Significant variations in the biochemical fingerprint of EVs were detected, with an elevation in peaks associated with lipid and protein signatures during early myogenic differentiation (day 2). Principal component analysis revealed a clear separation between the spectra of EVs derived from myogenic and senescent cell types, with non-overlapping interquartile ranges and population median. Observations aligned with nanoparticle tracking data, highlighting a significant early reduction in EV concentration in senescent myoblast cultures as well as notable variations in EV morphology and diameter. As differentiation progressed physical and biochemical differences in the properties of EVs became less pronounced. This study demonstrates the applicability of RS as a high-resolution analytical method for profiling biochemical changes in EVs during early myogenesis.