Drugs currently in use against Leishmania and Trypanosoma infections have limitations in terms of efficacy, safety, duration of treatment, toxicity, and resistance. It is therefore mandatory to identify molecular targets to be specifically inhibited. Folate is an essential cofactor in the biosynthesis of DNA and amino acids. The inhibition of its metabolism leads to alterations of cell replication and function. Only a few trypanosomatid enzymes of the folate pathway are presently discussed as potential targets, among them the bifunctional enzyme dihydrofolate reductase‐thymidylate synthase (DHFR‐TS) and pteridine reductase (PTR1). The identification of a specific enzyme such as PTR1, able to reduce folates other than biopterins, allowed the understanding of the resistance of trypanosomatids against known anti‐folate drugs. In most cases only the inhibition of both enzymes, DHFR‐TS and PTR1, would fully arrest the pathway's metabolic function. The proposed combination therapy opens up a novel approach: repositioning of the well‐established anti‐folate strategy for the treatment of trypanosomatid diseases by the discovery of novel anti‐folates that complement the efficacy profile of known drugs. The present chapter compiles the existing medicinal chemistry approaches specifically targeting the folate pathway in trypanosomatids, in particular PTR1 and the DHFR activity of DHFR‐TS. It covers the structural biology of the targets, related computational studies, core structure synthesis, and biological inhibitor characterization.