Phosphoproteomics has revolutionised our ability to rapidly quantify thousands of phosphorylation sites. Here, we present our phosphoproteome integrations of various exercise modalities in human skeletal muscle with cell- and rodent-based models to identify functionally relevant phosphorylation. We further profiled various exercise mimetics in vitro and identified direct kinase:substrate relationships via whole cell lysate in vitro kinase assays. Finally, we identify exercise-regulated phosphorylation sites that modulate in vivo skeletal muscle function and promote protein:protein interactions via mutational analysis.