Metabolomics is a comprehensive analysis of small molecules, or metabolites, in a system. Metabolomics is a hypothesis-generating experiment and offers an unbiased analysis of cell metabolism that can aid in the understanding of fundamental biological processes. Metabolomics is widely and broadly applicable in the biological sciences and has been used to study gene function, elucidate mechanisms of drug action, develop novel therapeutics, and to better understand disease states. This dissertation presents studies aimed at developing and determining the suitability of a mass spectrometry based untargeted metabolomics platform. Four chapters of original research are presented in this dissertation. The first chapter describes the development of a liquid chromatography-quadrupole-time-of-flight mass spectrometry metabolomics platform and details the various steps involved in a typical metabolomics experiment including metabolite extraction/sample preparation, metabolite separation and data collection, data processing and statistical analysis, and metabolite identification. The second chapter applies the metabolomics platform to uncover the metabolic consequences of vitamin C deficiency in zebrafish, which, like humans cannot synthesize vitamin C and must acquire it through diet for survival. In addition to uncovering several metabolic changes in vitamin C deficient zebrafish previously reported in genetic animal models of vitamin C deficiency, we found evidence for increased purine nucleotide cycle activity. These results demonstrate the suitability of zebrafish for the study of dietary vitamin C deficiency and highlight the roles of vitamin C in energy metabolism. The third chapter describes a metabolomics driven effort to characterize the antiobesity effects and mechanisms of xanthohumol, a prenylated flavonoid found in hops. Based on a metabolomics analysis of plasma from fatty rats treated with xanthohumol, we measure the bioenergetic effects of xanthohumol on cells in culture and find that it is a general mitochondrial uncoupler. We hypothesize that it is through this mechanism that xanthohumol exerts its anti-obesity effects in vivo. The fourth chapter investigates the temporal metabolome changes that occur during adipocyte differentiation. Using time course metabolomics, we uncovered increases in several uncharacterized di- and tripeptides, presumably products of protein degradation. We then treated differentiating adipocytes with ¹⁸O labeled water and found incorporation of ¹⁸O into the peptides, confirming them as products of peptide or protein hydrolysis. In addition, H₂¹⁸O metabolomics revealed enhanced flux through the CDP-choline cycle and activation of glutaminolysis during adipocyte differentiation, highlighting the utility of ¹⁸O labeled water metabolomics to uncover alterations in metabolic pathways undetectable with a typical metabolomics experiment.