Abstract Every organism requires cellular energy metabolism to meet developmental needs. When cells change from a resting to a proliferative state, the metabolic pathways are reprogrammed to meet the demands of proliferation. Diseases such as cancer can occur when changes in the metabolic pathways support unwanted functions such as uncontrolled proliferation. Assays for analyzing metabolites and metabolic enzymes are needed to understand cellular metabolic requirements and energy network regulation under normal and disease conditions. We exploited the robustness, sensitivity and broad dynamic range of bioluminescence detection to develop assays for the key metabolites lactate, glucose, glutamate and glucose-6-phosphate. The technology involves using specific dehydrogenases to couple metabolite levels to NAD(P)H production. In the presence of NAD(P)H, a novel proluciferin is converted to a substrate for luciferase resulting in light signals proportional to the starting metabolite concentration. Samples (e.g. mammalian cells, culture media, tissues, 3D microtissues) are added to the metabolite detection reagent in a 1:1 ratio. An inactivation solution added to complex biological samples lowers the background and increases sensitivity by lysing the cells, inactivating cellular enzymes, and destroying endogenous NAD(P)H. The sensitivity (1-5 pmol/sample) and broad assay window (maximum signal-to-background > 100 fold) of the assays allows simultaneous detection of multiple metabolites from the same set of samples. The wide assay window allows detection of very small changes in metabolite concentration, an advantage over conventional fluorescent and colorimetric detection methods. The assays adapt to various formats (96, 384-well plates) and require no instrumentation other than a luminometer. The assays can be used to measure the levels of critical metabolites and to monitor the effects of inhibitors and activators of glycolysis. The shift from oxidative phosphorylation to glycolysis can be monitored by measuring lactate production and secretion as well as glucose consumption from the same set of samples. The assays easily multiplex with cell viability assays enabling analysis of drug effects on cell proliferation, cell metabolism and mitochondria function. Citation Format: Mary Sobol, Jolanta Vidugiriene, Donna Leippe, Gediminas Vidugiris, Wenhui Zhou, James Cali. Metabolite assays to illuminate cellular energy networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2015-1211