Exercise plays an essential physiological role in the regulation of acute and long-term muscle health, insulin sensitivity and whole body metabolic homeostasis. However, only a few signalling pathways and downstream substrates are known to trigger these beneficial effects. We performed the first mass spectrometry (MS)-based phosphoproteomic analysis of human skeletal muscle biopsies obtained from healthy male subjects before and after an acute bout of high-intensity bicycle exercise. We identified 1,004 phosphosites that were significantly regulated with acute exercise. More than 900 of these phosphosites have not previously been associated with exercise and the upstream kinase is unknown. Given the known therapeutic potential of the AMPK pathway, we performed two additional MS screens to specifically pinpoint novel AMPK substrates in the human muscle including: (1) global MS-based phosphoproteomic analysis of myotubes with pharmacological AMPK activation; and (2) a novel global AMPK in vitro kinase assay combined with targeted phosphopeptide quantification with data-independent acquisition MS. Integration of these data sets revealed a number of novel AMPK substrates including A-kinase anchor protein 1 (AKAP1) that was validated in vivo. Phosphosite mutations identified a novel role for AMPK-phosphorylation of AKAP1 in mitochondrial respiration. We next investigated the use of proteomics to identify substrates involved in the regulation of long-term muscle growth. Follistatin, a potent inhibitor of TGFbeta-signalling, regulates muscle hypertrophy through an incompletely understood mechanism. Proteomic analysis of mice muscle-specific and inducible adeno-associated virus (AAV)-mediated overexpression of follistatin was performed over a time-course. Numerous proteins not previously implicated in the regulation of muscle growth were identified including the E3-ubiquitin ligase, ASB2. AAV-mediated overexpression of ASB2 induced significant muscle atrophy highlighting a novel regulator of muscle mass. These data and developed methodology will serve as an invaluable resource for future studies of muscle physiology and the development of novel therapeutics.