Microorganisms depend on their ability to modulate their metabolic composition according to specific circumstances, such as different phases of the growth cycle and circadian rhythms, fluctuations in environmental conditions, as well as experimental perturbations. A thorough understanding of these metabolic adaptations requires the ability to comprehensively identify and quantify the metabolome of bacterial cells in different states. In this review, we present an overview of the diverse metabolomics approaches recently adopted to explore the metabolism of a wide variety of microorganisms. Focusing on a selection of illustrative case studies, we assess the different experimental designs used and explore the major achievements and remaining challenges in the field. We conclude by discussing the important complementary information provided by computational methods such as genome-scale metabolic modeling, which enable an integrated analysis of metabolic state changes in the context of overall cellular physiology. © 2011 Future Science Ltd.