Metabolomics approaches provide an analysis of changing metabolite levels in biological samples. In the past decade, technical advances have spurred the application of metabolomics in a variety of diverse research areas spanning basic, biomedical, and clinical sciences. In particular, improvements in instrumentation, data analysis software, and the development of metabolite databases have accelerated the measurement and identification of metabolites. Metabolomics approaches have been applied to a number of important problems, which include the discovery of biomarkers as well as mechanistic studies aimed at discovering metabolites or metabolic pathways that regulate cellular and physiological processes. By providing access to a portion of biomolecular space not covered by other profiling approaches (e.g., proteomics and genomics), metabolomics offers unique insights into small molecule regulation and signaling in biology. In the following review, we look at the integration of metabolomics approaches in different areas of basic and biomedical research, and try to point out the areas in which these approaches have enriched our understanding of cellular and physiological biology, especially within the context of pathways linked to disease.