Summary Tuberculosis remains a major global health burden with 10 million new cases in 2019 and a latently-infected population of billions. Deaths from tuberculosis now exceed those from HIV (1.2 million in 2019). Mycobacterium tuberculosis, the causative agent, is a sophisticated pathogen which can persist for decades in the human host and which requires lengthy treatment for cure with multiple antibiotics. One of the features of M. tuberculosis is its ability to survive and replicate inside human cells, including macrophages, one of the normal host defense mechanisms against infection. Intracellular bacteria are a specific population which can be hard to kill, in part due to the requirement that molecules enter eukaryotic cells, and in part due to the different physiological state in which the bacteria persist. Increasing evidence points to a higher level of antibiotic tolerance in intracellular bacteria, as well as increased heterogeneity. In order to find new agents, we developed a phenotypic screening method utilizing high content screening to monitor bacterial and eukaryotic cell survival simultaneously. We ran a pilot screen to identify novel chemical inhibitors of mycobacterial intracellular replication. We found several series of interest and selected three series with attractive physicochemical properties. Based on our initial success we propose to expand our effort to run a larger diverse screening set. The overall aim of this proposal is to screen a diversity set of molecules against intracellular Mtb and to evaluate and prioritize hits for future work. We will combine biological activity, profile, mode of action and physicochemical properties to select series with the most promise. The major outcome of this exploratory proposal will be novel chemical matter ready to enter the discovery pipeline.