Genome-wide predictions of microbial metabolic networks are commonly used to predict the druggable space of different microbial pathogens. However, many microbial genes remain functionally undefined highlighting potential gaps in these networks. Furthermore, recent high mass resolution mass spectrometry metabolite profiling studies have highlighted the presence of large numbers of chemically undefined metabolite peaks in microbial extracts. Undefined metabolite peaks typically account for more than half of all detected peaks, indicating that many metabolic processes and potential drug targets remain to be discovered. We have developed a new approach for defining the 'dark metabolome' of microbial pathogens, that involves parallel metabolic labelling with a panel of 13C-labeled carbon sources followed by detection of labeled metabolites using complementary mass spectrometry platforms. These analyses have been used to identify a number of novel metabolites and potential pathways in different pathogens. A significant number of ‘new’ metabolites are likely by-products of enzymes in central carbon metabolism. These metabolites can be allosterically active or toxic if they accumulate and often need to be catabolized by 'metabolite repair enzymes. These analyses suggest that characterization of the dark metabolome will reveal processes that underpin the evolution of new pathways, as well as highlighting novel drug targets.