This protocol describes the combined use of metabolite profiling and stable isotope labelling to define pathways of central carbon metabolism in the protozoa parasite, Leishmania mexicana. Parasite stages are cultivated in standard or completely defined media and then rapidly transferred to chemically equivalent media containing a single (13)C-labelled nutrient. The incorporation of label can be followed over time or after establishment of isotopic equilibrium by harvesting parasites with rapid metabolic quenching. (13)C enrichment of multiple intracellular polar and apolar (lipidic) metabolites can be quantified using gas chromatography-mass spectrometry (GC-MS), while the uptake and secretion of (13)C-labelled metabolites can be measured by (13)C-NMR. Analysis of the mass isotopomer distribution of key metabolites provides information on pathway structure, while analysis of labelling kinetics can be used to infer metabolic fluxes. This protocol is exemplified using L. mexicana labelled with (13)C-U-glucose. The method can be used to measure perturbations in parasite metabolism induced by drug inhibition or genetic manipulation of enzyme levels and is broadly applicable to any cultured parasite stages.