Comparative metabolic profiling of cancerous and normal cells improves our understanding of the fundamental mechanisms of tumorigenesis and opens new opportunities in target and drug discovery. Here we report a novel methodology of comparative metabolome analysis integrating the information about both metabolite pools and fluxes associated with a large number of key metabolic pathways in model cancer and normal cell lines. The data were acquired using [U-13C]glucose labeling followed by two-dimensional NMR and GC-MS techniques and analyzed using isotopomer modeling approach. Significant differences revealed between breast cancer and normal human mammary epithelial cell lines are consistent with previously reported phenomena such as upregulation of fatty acid synthesis. Additional changes established for the first time in this study expand a remarkable picture of global metabolic rewiring associated with tumorigenesis and point to new potential diagnostic and therapeutic targets.