The exponential growth in the number of sequenced microorganisms versus the relative slow influx of direct biochemical characterization of microbes is limiting the utility of sequence information. High-throughput experimental approaches to functionally characterize microbial metabolism are urgently needed to leverage genome sequences for example: to understand host-microbe interactions, microbial communities, to utilize microbes for bioenergy, bioremediation, etc. Mass spectrometry based small molecule metabolite analysis is rapidly becoming a method of choice to meet these needs and enables multiple paths to discovering and validating the functional assignments. Approaches range from the targeted in vitro screening of small sets of metabolic transformations to define enzymatic activities to global metabolic profiling (metabolomics) to define metabolic pathways and gain insights into microbial responses to environmental and genetic perturbations. The combination of metabolite profiling with genome-scale models of metabolism and other -omic approaches provides opportunities to expand our understanding of microbial metabolic networks, stress responses, and to identify genes associated with specific enzymatic and regulatory activities.