Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.