Until recently, 13C-based flux analyses have almost exclusively relied on analysis of labelled amino acids in proteins. This approach is not directly applicable to Leishmania, as these parasites scavenge most of their amino acids from the media. Leishmania are also unusual in that they i) share little genomic similarity with other organisms ii) constitutively express their metabolic genes and iii) display minimal changes in the enzyme levels throughout their life cycle stages. The three factors have contributed to an early development of comprehensive and reproducible 13C-based metabolomics approaches in these parasites. The work presented here contributes to the creation of new 13C-based metabolic flux approaches based on the isotopologue analysis of free metabolite pools in Leishmania mexicana. Namely, a new approach is presented for simultaneous calculation of in vivo fractional fluxes (or flux ratios) into two or more metabolite nodes with carbon dioxide condensation, based on isotopologue analysis of free metabolite pools. This method is used to perform the first quantitative in vivo fractional flux calculation of central carbon metabolism in any human parasite.