Despite extensive efforts in tuberculosis (TB) drug research, very few novel inhibitors have been discovered. This issue emphasizes the need for innovative methods to discover new anti-TB drugs. In this study, we established a new high-throughput screen (HTS) platform technology that differs from traditional TB drug screens because it utilizes Mycobacterial-Protein Fragment Complementation (M-PFC) to identify small molecule inhibitors of protein-protein interactions in mycobacteria. Several examples of protein-protein interactions were tested with M-PFC to highlight the diversity of selectable drug targets that could be used for screening. These included interactions of essential regulators (IdeR dimerization), enzymatic complexes (LeuCD), secretory antigens (Cfp10-Esat6), and signaling pathways (DevR dimerization). The feasibility of M-PFC in a HTS platform setting was tested by performing a proof-of-concept quantitative HTS of 3,600 small molecule compounds on DevR-DevR interaction, which was chosen because of its strong implications in Mycobacterium tuberculosis persistence and the need for effective drugs against latent TB. The calculated Z′-factor was consistently ≥0.8, indicating a robust and reproducible assay. Completion of the proof-of-concept screen allowed for the identification of advantages and disadvantages in the current assay design, where improvements made will further pioneer M-PFC-based applications in a large-scale HTS format. © 2011, Mary Ann Liebert, Inc.