Myogenesis is governed by signalling networks whose regulations are tightly controlled in a time-dependent manner. While different protein kinases have been identified to regulate various aspects of myogenesis, knowledge on the global signalling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using mass spectrometry (MS)-based phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict substrates of key kinases that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signalling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles on myogenesis.