Through the characterization of metabolic pathways, metabolomics is able to illuminate the activities of a cell at the functional level. However, the metabolome, which is comprised of hundreds of chemically diverse metabolites, is rather difficult to monitor. Mass spectrometry (MS)-based profiling methods are increasingly being utilized for this purpose. To this end, MS is often coupled to the separation techniques gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). The mass-based selectivity that the MS provides, together with the chromatographic or electrophoretic separation of analytes, creates hyphenated techniques that are ideally suited to the measurement of large numbers of metabolites from microbial extracts. In this chapter, we describe GC-MS, LC-MS, and CE-MS methods that are applicable to microbial metabolomics experiments.