Mouse-derived C2C12 myoblasts serve as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocyte formation comprehensively, we used large scale gel-free tandem mass spectrometry to monitor global proteome alterations throughout a time course analysis of the myogenic C2C12 differentiation program. The relative abundance of approximately 1,800 high confidence proteins was tracked across multiple time points using capillary scale multidimensional liquid chromatography coupled to high throughput shotgun sequencing. Hierarchical clustering of the resulting profiles revealed differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across the early, mid, and late stages of differentiation. Several hundred previously uncharacterized proteins were likewise detected in a stage-specific manner, suggesting novel roles in myogenesis and/or muscle function. These proteomic data are complementary to recent microarray-based studies of gene expression patterns in developing myotubes and provide a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.