Metabolomics enables the comprehensive profiling of cellular metabolites at the systems level, thereby providing a direct readout of biochemical activity that can be correlated with phenotype and used to identify therapeutic targets. Although several challenges remain to be addressed, emerging mass spectrometric and bioinformatic technologies have already proven to be effective tools for diagnostics and for providing insights into cell metabolism. Metabolites, the chemical entities that are transformed during metabolism, provide a functional readout of cellular biochemistry. With emerging technologies in mass spectrometry, thousands of metabolites can now be quantitatively measured from minimal amounts of biological material, which has thereby enabled systems-level analyses. By performing global metabolite profiling, also known as untargeted metabolomics, new discoveries linking cellular pathways to biological mechanism are being revealed and are shaping our understanding of cell biology, physiology and medicine.