Mycobacterium tuberculosis(Mtb) is the etiological agent of tuberculosis (TB), a disease that, although preventable and curable, remains a global epidemic due to the emergence of resistance and a latent form responsible for a long period of treatment. Drug discovery in TB is a challenging task due to the heterogeneity of the disease, the emergence of resistance, and uncomplete knowledge of the pathophysiology of the disease. The limited permeability of the cell wall and the presence of multiple efflux pumps remain a major barrier to achieve effective intracellular drug accumulation. While the complete genome sequence of Mtb has been determined and several potential protein targets have been validated, the lack of adequate models for in vitro and in vivo studies is a limiting factor in TB drug discovery programs. In current therapeutic regimens, less than 0.5% of bacterial proteins are targeted during the biosynthesis of the cell wall and the energetic metabolism of two of the most important processes exploited for TB chemotherapeutics. This review provides an overview on the current challenges in TB drug discovery and emerging Mtb druggable proteins, and explains how chemical probes for protein profiling enabled the identification of new targets and biomarkers, paving the way to disruptive therapeutic regimens and diagnostic tools.