Even though they account for 10% of the global disease burden and besides the new record of funding in 2018, tropical diseases are still neglected by the majority of pharmaceutical companies and public funding. The reason can be mostly found in the fact that for these diseases a conventional drug discovery process is often too expensive. The development of new approaches in the early stage of drug discovery has therefore a key role in fighting Neglected Tropical Diseases (NTD). AEGIS is a European network which relates on collaborations for a multidisciplinary approach, in order to “accelerate” drugs development – hence reducing the costs. As part of the network, this project is focused on a rational exploration of the chemical space, together with an in depth-analysis of molecular interactions for a better characterization of targets involved in NTD. One cost-efficient way for exploring pharmacologically relevant chemical space is fragment based lead discovery (FBLD). This approach requires an extensive understanding of the target properties; therefore, a pipeline of orthogonal methods was developed to validate the suitability of the target for FBLD. The choice of a fragment library has a significant impact on the experimental strategy. Not only the validation of the target, but also practical issues concerning technology, orthogonal validation and applicability have to be considered when initiating a fragment screening campaign. Another rational approach for the discovery of new drugs is looking at the target structure, especially when considering protein complexes interaction. Through the structural analysis of the protein-protein interface, several short peptides derived from the binding partner were analysed in their interaction with the target both in vitro and in cell. This allowed to identify the key sequence for the binding to the target and the internalization of the complex, both crucial information for a structure-based approach. The internalization process of the target was characterized by a real-time cell binding assay (RT-CBA), revealing a higher level of complexity than what was previously described. Implementing RT-CBA in the early stage is a strategy that might improve the success rate of drug development. The possibility to develop an intracellular time resolved molecular interaction assay between small molecules and a model target fused to a fluorescent protein was therefore explored. From a fragment screening campaign to a structure-based approach, culminating with a real time intracellular validation of hits, all the assays developed address the different possibilities for accelerating the drug discovery process in the early stages.