Enzymes fuel the biochemical activities of all cells. Their substrates and products thus represent a potential window into the physiologic state of a cell. Metabolomics focuses on the global, or systems-level, study of small molecules in a given biological system and has thus provided an experimental tool with which to study cellular physiology, including the biochemistry within pathogenic microorganisms. While metabolomic studies of Mycobacterium tuberculosis are still in their infancy, recent studies have begun to deliver unique insights into the composition, organization, activity, and regulation of the bacterium's physiologic network not accessible by other approaches. Here, we outline practical methods for the culture, collection, and analysis of metabolomic samples from M. tuberculosis that emphasize minimally perturbing sample handling, broad and native metabolite recovery, and sensitive, biologically agnostic metabolite detection.