Multiple kinase activities are required for skeletal muscle differentiation. However the mechanisms by which these kinase pathways converge to co-ordinate the myogenic process is unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determined specific peptide sequences using LC-MS/MS. To place these phosphopeptides into a rational context, a bioinformatic approach was used. Phosphorylation sites were matched to known site-specific and to non site-specific kinase-substrate interactions, then other substrates and upstream regulators of the implicated kinases were incorporated into a model network of protein-protein interactions. The model network implicated several kinases of known relevance to myogenesis including Akt, Gsk3, CDK5, p38, Dyrk and MAPKAPK2 kinases. This combination or proteomics and bioinformatics technologies should offer great utility as the volume of protein-protein and kinase-substrate information continues to increase.