ABSTRACT Nonreplicating or dormant cells of Mycobacterium tuberculosis constitute a challenge to tuberculosis (TB) therapy because of their tolerance or phenotypic resistance to most drugs. Here, we propose a simple model for testing drugs against nongrowing cells that exploits the 18b strain of M. tuberculosis , a streptomycin (STR)-dependent mutant. Optimal conditions were established that allowed 18b cells to replicate in the presence of STR and to survive, but not multiply, following withdrawal of STR. In the presence of the antibiotic, M. tuberculosis 18b was susceptible to the currently approved TB drugs, isoniazid (INH) and rifampin (RIF), and to the experimental drugs TMC207, PA-824, meropenem (MER), benzothiazinone (BTZ), and moxifloxacin (MOXI). After STR depletion, the strain displayed greatly reduced susceptibility to the cell wall inhibitors INH and BTZ but showed increased susceptibility to RIF and PA-824, while MOXI and MER appeared equipotent under both conditions. The same potency ranking was found against nonreplicating M. tuberculosis 18b after in vivo treatment of chronically infected mice with five of these drugs. Despite the growth arrest, strain 18b retains significant metabolic activity in vitro , remaining positive in the resazurin reduction assay. Upon adaption to a 96-well format, this assay was shown to be suitable for high-throughput screening with strain 18b to find new inhibitors of dormant M. tuberculosis .