Selection of appropriate targets for launching antituberculosis drug discovery programmes is challenging. This challenge is magnified by the limited repertoire of ‘validated targets’ and the paucity of clinically successful drugs. However, continued understanding of the biology of the microbe and its interaction with the host has enabled detailed evaluation of several interesting pathways and novel targets. The value of a target that is suitable for antituberculosis drug discovery needs to be defined not only in the context of its ‘essentiality’ for survival in vitro but also against a variety of properties relevant to activities in the drug discovery process, e.g.; selectivity, vulnerability, suitability for structural studies, ability to monitor inhibition in whole cells etc. It is also rarely feasible to obtain all the relevant information on the target prior to the launch of a discovery programme. Thus, there is a continuous confidencebuilding exercise on the validity of a target. Several novel approaches have enabled exploitation of the mycobacterial genome and prioritisation of putative targets; the concept of ‘sterilisation’ is now being evaluated not only through the availability of structurally diverse probe compounds but also by the ability to characterise metabolic pathways in vivo. The impact of the current knowledge base on the different facets of ‘target validation’ relevant to antituberculosis drug discovery is discussed in this article with emphasis on developing appropriate matrix systems to prioritise them. The article also discusses the influence of lead generation approaches with specific reference to antibacterial drug discovery.