Protozoan parasites are an evolutionarily diverse group of single-celled eukaryotes that cause a number of important diseases, such as malaria, toxoplasmosis and leishmaniasis, that collectively affect hundreds of millions of people world-wide. There are no vaccines for any of these diseases and current drug therapies are extremely limited and/or being undermined by the emergence of drug-resistant parasite lines, and there is a urgent need to identify new drug targets. While metabolic enzymes are high priority drug targets, our current understanding of the cellular metabolism and physiology of intracellular parasite stages is limited. In particular, while genome-wide annotations have provided broad insights into the metabolic potential of these pathogens, the majority of protein-encoding genes in these genomes (60-70%) have no known function. Furthermore, attempts to infer stage-specific changes in metabolism from transcriptional or proteomic analyses have also been hampered by the reliance of these organisms on post-translational responses. In order to identify metabolic pathways that are active in pathogenic stages we have developed a comprehensive suite of MS-based metabolomic approaches for characterizing parasite metabolism in situ. In this talk, examples will be provided of how MS-based metabolite profiling coupled with ¹³C-stable isotope labelling approaches have revealed novel aspects of parasite metabolism that are essential for intracellular survival. We have also developed new approaches for measuring parasite and host responses in infected animal tissues using heavy water (²H₂O) labelling and high resolution FTICR-imaging mass spectrometry. The latter approaches are being used to identify metabolically distinct populations of pathogens in tissues that may be differentially susceptible to anti-parasite drugs.