© 2013 Springer Science+Business Media, LLC. All rights are reserved. An important challenge for TB investigators in the postgenomic era is to integrate distinct functional strategies to study the molecular mechanism of Mycobacterium tuberculosis (Mtb) virulence. However, the biological function of the majority of Mtb genes is unknown. This has revealed the need for an approach to convert raw genome sequence data into functional information. In the past decade, the yeast two-hybrid system (Y2H) has contributed significantly towards studying TB virulence and persistence, but has several drawbacks. Recently, several mycobacterial protein-protein interaction (PPI) technologies have been reported that helped propose functions for unknown proteins through guilt by association and will be discussed in this chapter. We will examine the advantages, disadvantages and limitations of these systems and how these technologies can be used to dissect-signaling, drug resistance, and virulence pathways. We will also discuss how-mycobacterial PPI technologies can be exploited to force proteins to interact and for the discovery of small-molecule inhibitors against protein complexes. In sum, by characterizing Mtb PPIs on a genomic scale, it will be possible to assemble-physiologically relevant protein pathways in mycobacteria, the outcome of which will be invaluable for determining virulence mechanisms and the function of previously uncharacterized proteins.