Trypanosoma cruzi is the causal agent of American Trypanosomiasis or Chagas Disease in humans. The current drugs for its treatment benznidazole and nifurtimox have inconveniences of toxicity and efficacy; therefore, the search for new therapies continues. Validation through genetic strategies of new drug targets against the parasite metabolism have identified numerous essential genes. Target validation can be further narrowed by applying Metabolic Control Analysis (MCA) to determine the flux control coefficients of the pathway enzymes. That coefficient is a quantitative value that represents the degree in which an enzyme/transporter determines the flux of a metabolic pathway; those with the highest coefficients can be promising drug targets. Previous studies have demonstrated that cysteine (Cys) is a key precursor for the synthesis of trypanothione, the main antioxidant metabolite in the parasite. In this research, MCA was applied in an ex vivo system to the enzymes of the reverse transsulfuration pathway (RTP) for Cys synthesis composed by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL). The results indicated that CGL has 90% of the control of the pathway flux. Inhibition of CGL with propargylglycine (PAG) decreased the levels of Cys and trypanothione and depleted those of glutathione in epimastigotes (proliferative stage in the insect vector); these metabolite changes were prevented by supplementing with Cys, suggesting a compensatory role of the Cys transport (CysT). Indeed, Cys supplementation (but not PAG treatment) increased the activity of the CysT in epimastigotes whereas in trypomastigotes (infective stage in mammals) CysT was increased when they were incubated with PAG. Our results suggested that CGL could be a potential drug target given its high control on the RTP flux and its effects on the parasite antioxidant defense. However, the redundant Cys supply pathways in the parasite may require inhibition of the CysT as well. Our findings also suggest differential responses of the Cys supply pathways in different parasite stages.