Metabolic profiling of cell line collections has become an invaluable tool to study disease etiology, drug modes of action and to select personalized treatments. However, large-scale in vitro dynamic metabolic profiling is limited by time-consuming sampling and complex measurement procedures. By adapting a mass spectrometry-based metabolomics workflow for high-throughput profiling of diverse adherent mammalian cells, we establish a framework for the rapid measurement and analysis of drug-induced dynamic changes in intracellular metabolites. This methodology is scalable to large compound libraries and is here applied to study the mechanism underlying the toxic effect of dichloroacetate in ovarian cancer cell lines. System-level analysis of the metabolic responses revealed a key and unexpected role of CoA biosynthesis in dichloroacetate toxicity and the more general importance of CoA homeostasis across diverse human cell lines. The herein-proposed strategy for high-content drug metabolic profiling is complementary to other molecular profiling techniques, opening new scientific and drug-discovery opportunities.