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Visceral leishmaniasis (VL) and human African trypanosomiasis (HAT) are tropical neglected diseases caused by the respective parasites, Leishmania infantum or L. dononani and Trypanosoma brucei gambiense or T. b. rhodesiense. These parasite species can cause serious infections resulting in serious clinical symptoms and eventual death when left untreated. Current treatment options for VL and HAT are less than adequate as they lack efficacy, are not devoid of adverse effects, and suffer from an increasing incidence of treatment failure. Hence, new drugs are needed, and multiple efforts have recently resulted in the identification of nitroimidazoles, oxaboroles and aminopyrazoles as promising antileishmanial lead series and of pyrazolopyrimidinones as a potential class of antitrypanosomal compounds. The first aim was to evaluate the antiparasitic effects of these novel compounds to assess their further potential in the field. The novel antileishmanial lead series were shown to have killing potential both in vitro and in vivo, and were additionally effective in strains resistant to current antileishmanial drugs. The excellent in vitro activity of antitrypanosomal pyrazolopyrimidinones did not translate in mouse models, with inadequate parasite clearance in an acute first-stage infection model and with additional toxicity issues in the chronic second-stage disease model. Improvement of this lead series is necessary before considering further development. The second aim was to determine the mechanism-of-action (MoA) and mode-of-resistance (MoR) of the selected lead series. A number of techniques was used to unravel the drug target of the antileishmanials, including resistance selection followed by whole-genome sequencing, selection of a genome-wide overexpression library (Cosmid-sequencing), metabolomics, and an upcoming proteomics technique, named drug affinity responsive target stability (DARTS). These did not result in the unequivocal deconvolution of the drug target but identified multiple genes putatively involved in the MoA and MoR of the drugs. The MoA of the pyrazolopyrimidinones, on the other hand, was studied using a whole-genome knockdown library combined with an untargeted metabolomics approach. The S-adenosylmethionine (AdoMet) pathway was found to be commonly perturbed, suggesting its involvement in the MoA of the compound. Single gene knockdown and overexpression experiments could not yet identify the drug target. To conclude, it is clear that the nitroimidazoles, aminopyrazoles and oxaboroles are very promising and could become the next antileishmanial drugs on the market. Rapid resistance development in the field might not be a primary issue and implementation of these drugs in combination regimens might be possible as the MoA of each series is clearly distinct and differs from the conventional antileishmanial drugs. However, the MoA and MoR of these series need to be further explored with complementary techniques. The pyrazolopyrimidinones, on the contrary, need to be further optimized to achieve an enhanced efficacy in the two stages of HAT disease. The identification of the AdoMet pathway as potential drug target may help in the discovery and synthesis of more potent and safer alternatives.