Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the leading cause of death amongst bacterial infections worldwide, with drug resistance strains potentially killing more than 10 million people by 2050. TB infection is associated with poverty and poor healthcare access in low-income counties; note therefore that drug development can only contribute to its final eradication only if access to medicines is improved. The identification of the mechanism of action of hit and lead molecules, in vivo efficacy and toxicity issues are the biggest bottlenecks observed in drug discovery. Therefore, the validation of new pathways targeted by novel chemical entities is essential to ensure success in the early stages of drug discovery. In this context, the aspartyl-tRNA synthetase of M.tb (Mt-AspS) represents an attractive target since very few approved inhibitors target aminoacyl-tRNA synthesis, decreasing the likelihood of drug resistance. We have identified a number of inhibitors in a phenotypic screen that have been further validated in several biochemical assays and investigated the chemical space around these molecules through a structure activity relationship (SAR) map. The results of my thesis will set out future guidelines for the discovery of novel protein synthesis inhibitors against the other eighteen tRNA-synthetases of the M.tb proteome.