AbstractAlong with genes and proteins, metabolites play important roles in sustaining life. Their functions include “primary” functions in metabolism and energy storage, as well as “secondary” functions in cell‐to‐cell signaling, metal acquisition, and virulence. There remains much to be learned about the in vivo roles of metabolites. Approaches that accelerate measurement of metabolite levels directly from cells and tissues should increase our understanding of the diverse roles of metabolites and potentially lead to discovery of novel metabolites and metabolic pathways. Metabolomics is an important comparative tool to study global metabolite levels in samples under various conditions. In this unit, the steps needed to perform a mass spectrometry (MS)–based untargeted metabolomics experiment using bacterial supernatants are detailed. In contrast to a targeted metabolomics experiment, which measures ions from known metabolites, an untargeted metabolomics experiment registers all ions within a certain mass range, including ions belonging to structurally novel metabolites. The protocols in this unit describe the conditions necessary for analyzing hydrophobic metabolites and provide an example of how to structurally characterize a novel metabolite. Curr. Protoc. Mol. Biol. 90:30.1.1‐30.1.24. © 2010 by John Wiley & Sons, Inc.