Pyrazinamide (PZA) is a component of front-line chemotherapy against Mycobacterium tuberculosis. Its inclusion has shortened treatment to 6-months due to its ability to act against non-replicating/slow-growing bacilli. M. tuberculosis encounters acidic environments during infection, including the granuloma, where PZA is active. PZA has no early bactericidal activity during treatment and assumptions have been made that PZA has limited activity against fast-growing bacteria. Can PZA activity be extended to fast-growing bacilli, thereby further shortening treatment times? There are challenges in assessing PZA activity in vitro, as the antibiotic requires low acidity to be active, and this is technically difficult to control and avoided because of a lack of standardisation. The optimal acidity for PZA activity (pH 5.6) is too low for bacterial growth. Assays that allow acidity levels low enough for PZA activity and allow for growth of M. tuberculosis would enable the assessment of PZA activity against actively dividing bacteria. We aimed to determine the growth conditions under which PZA could be active against dividing M. tuberculosis, using defined controlled conditions in chemostat culture and found that PZA was indeed active against fast growers under acidic conditions. We subsequently asked whether PZA can be boosted by potentiators. We developed a reproducible, low-pH plate assay to identify compounds that would boost the activity of PZA against dividing bacilli. Compound libraries were screened through a Tres Cantos Openlab Foundation-funded project and hit compounds are being studied further to determine their modes of action, some of which target the electron transport chain.