Objective To establish a rapid adenosine triphosphate ( ATP) bioluminescence assay for assessing drug resistance of Mycobacterium tuberculosis( M. tuberculosis) . Methods In this study mycobacterial growth was monitored using a bioluminescence assay of mycobacterial ATP. Cultures of M. tuberculosis H37Rv and of 10 clinical isolates of the same species were exposed to serial dilutions of rifampicin. Results Suppression of ATP, which indicates growth inhibition, occurred for susceptible but not for resistant strains, within five to seven days of incubation. Breakpoint concentrations between susceptible and resistant strains were determined by comparing these results with those obtained by the reference method ( BACTEC 3D) . Full agreement was found in 100% of the assays with the resistance ratio method on Lowenstein-Jensen ( L-J) medium, and 100% of the assays were in full agreement with the radiometric system( BACTEC 3D) . Conclusion The bioluminescence assay of mycobacterial ATP has comparable ability to detect drug resistant strains of M. tuberculosis; A main advantage of the bioluminescence method is its rapidity; Results are available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium.