The African Trypanosomiases, also commonly called African sleeping sickness in humans (HAT) and “Nagana” in domestic livestock, are fatal neglected diseases that occur in 36 sub-Saharan African countries. The disease progresses through two stages and is caused by two subspecies of the parasite T. brucei: T. b. gambiense (West Africa; Tbg) and T. b. rhodesiense (East Africa; Tbr). T. brucei is also pathogenic to wild and domestic animals causing “Nagana”, a disease that has a significant impact on socioeconomic development in many parts of rural Africa. Current treatments are considered unsatisfactory due to treatment failures and high toxicity. Therefore, there is a great need of new and cost-effective drugs to treat the disease, especially at later stages when the parasites infect the brain. Drug discovery efforts are nowadays directed towards natural products and medicinal plants represent a validated source for discovery of new lead compounds and standardized herbal medicines against trypanosomiases [1]. Tithonia diversifolia (Hemsl.) A.Gray, well-known as Mexican sunflower, is a bushy perennial weed commonly found on the fields, wasteland and road sides of tropical areas in South America, Asia and Africa. The plant belongs to the Asteraceae family and used as traditional medicine for the treatment of various diseases, including malaria [2]. The phytochemical analyses of T. diversifolia indicate1d the presence of bioactive substances such as alkaloids, saponins, glycosides, flavonoid, tannins, terpenoid and phenols in the methanolic extract [3]. The leaves methanolic extract showed a quite remarkable inhibitory activity against Trypanosoma brucei brucei TC221. For this reason, a chromatographic separation of total methanolic extract has been performed, obtaining 17 fractions. The phytochemical composition of crude extract and purified fractions were investigated using HPLCESI- MS/MS and 1D and 2D NMR spectra. The isolated fractions have been selected as valid candidates for investigation as potential inhibitors of T. brucei. The results of this study will be discussed.