AbstractArginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many carcinomas and in the majority of sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing ASS1. As arginine-based multiagent therapies are being developed, further characterization of the changes induced by arginine starvation is needed. In order to develop a systems-level understanding of these changes, activity-based proteomic profiling (ABPP) and phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-PEG20-sensitive and resistant sarcoma cells. When integrated with metabolomic profiling, this multi-omic analysis reveals that cellular response to arginine starvation is mediated by adaptive ERK signaling and activation of the Myc–Max transcriptional network. Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by enhancing glutamine and pyruvate anaplerosis and altering lipid metabolism to recycle citrate for oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, these multi-omic approaches could provide valuable tools for evaluating response to metabolically targeted therapies.