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Novel drugs and improved diagnostics for Mycobacterium tuberculosis (MTB) are urgently needed and go hand in hand. We evaluated the in vitro activity of two benzothiazinone drug candidates (MCZ, PBTZ169; BTZ043) and their main metabolites against MTB using advanced nanomotion technology. The results demonstrated significant reductions in MTB viability within 7 h, indicating the potential for rapid, precise antibiotic susceptibility testing based on a phenotypic read-out in real time. PBTZ169 and H 2 -PBTZ169 achieved 100% separation between the susceptible H37Rv and a resistant dprE1 mutant strain NTB1. These findings support nanomotion technology's potential for faster antibiotic susceptibility testing of novel MTB drug candidates targeting the DprE1 enzyme that could reduce empirical treatment duration and antibiotic resistance selection pressure due to inaccurate treatments., Competing Interests: D. Cichocka, A. Luraschi-Eggemann, G. Cathomen, and A. Sturm are employed by Resistell. A. Vocat is employed at the Institute of Microbiology of the University of Lausanne and Resistell AG. G. Greub is a medical advisor for Resistell AG.