Mycobacterium tuberculosis, the causative agent of tuberculosis claims about five thousand lives daily world-wide, while one-third of the world is infected with latent tuberculosis. The emergence of multidrug-resistant M. tuberculosis strains has imposed an urgent call for antibiotics with novel mechanisms of action. A target-based approach has been taken to address this pressing issue, by identifying potent and selective inhibitors of methionine aminopeptidase (MetAP) from M. tuberculosis. MetAP is a metalloprotease that removes the N-terminal methionine during protein synthesis. The essential role of MetAP in Escherichia coli and other microorganisms makes it a promising target for the development of new antibiotics. Therefore, two MetAPs, MtMetAP1a and MtMetAP1c from M. tuberculosis, have been cloned, overexpressed and purified to near homogeneity. Using a high-throughput screening approach with a coupled methionine-proline aminopeptidase assay, over 175,000 compounds have been screened against MtMetAP1c and potent inhibitors have been identified that have activity against replicating and dormant M. tuberculosis. A combination of chemical and genetic approaches was then used to validate MtMetAPs as viable targets. First, it was found that over-expressing either MtMetAP1a or MtMetAP1c in M. tuberculosis in the presence of inhibitor resulted in a gain in resistance in comparison to the wild-type strains. In addition, the essentiality of both MtMetAPs for growth and survival of M. tuberculosis was determined using knock-down strains of MtMetAP1a and MtMetAP1c. The results from the colony counts after three weeks showed that knockdown of MtMetAP1a resulted in decreased viability of 76.0% in M. tuberculosis. These results suggest that both MtMetAP1a and Mt MetAP1c are promising anti-tuberculosis targets. Furthermore, the crystal structure of MtMetAP1c has been solved in collaboration with Dr. Brian Matthews at the University of Oregon. This will facilitate the rational design and synthesis of more potent and selective inhibitors of MtMetAPs as novel leads for anti-tuberculosis drug development.