ABSTRACT Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo . In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens . Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro . Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.