Exercise improves health through adaptive metabolic and mechanical remodelling, mediated by a network of kinases in response to homeostatic stress. A comprehensive understanding of these signalling pathways will translate into improved strategies for the development of exercise mimetics to treat a range of diseases. Consequently, an in vitro model of exercise is required to delineate these signalling networks, kinase-substrate relationships and interrogate functional phosphorylation. We utilised a novel high-throughput phosphoproteomic screen to measure the action and interaction of candidate exercise-like treatments in rat L6 myotubes. Exercise-like treatments were selected if they activate a known or potential exercise kinase, or provide a stress similar to exercise. A total of 21 exercise-like treatments were initially screened and subsequent phosphoproteomic analysis using single-shot LFQ was performed on a total of 67 individual samples (10 treatments including paired controls, n=3-5). This quantified 20,249 unique Class I phosphopeptides, of which 37.8% were regulated in at least one treatment. Statistical and comparative analysis revealed the treatments regulate diverse modules in the phosphoproteome. Kinase-level analysis demonstrated drug action and provided a resource for future in vitro kinase-substrate and functional studies. Cross-species mapping revealed that 74% of the regulated phosphosites in exercised human skeletal muscle that mapped to the rat experiment were regulated in at least one treatment. Through enrichment analysis and a novel scoring system, it is evident that isoproterenol, thapsigargin and the combination of isoproterenol and berberine most closely recapitulate the exercise phosphoproteome. The in vitro combination of isoproterenol and berberine demonstrated the extent of interactions between candidate exercise-like treatments, as >200 phosphopeptides were uniquely regulated by the combination. This reveals the importance of considering drug interactions, given the range of stimuli involved in physiological processes, such as exercise.