Background. It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis. Methods. To assess treatment response, sputum samples (n = 124) from unselected patients (n = 35) with sputum microscopy-positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric first-line therapy. Tuberculosis quantitative viability microscopy with fluorescein diacetate, quantitative culture, and acid-fast auramine microscopy were all performed in triplicate. Results. Tuberculosis quantitative viability microscopy predicted quantitative culture results such that 76% of results agreed within ±1 logarithm (r S = 0.85; P <.0001). In 31 patients with non-multidrug-resistant (MDR) tuberculosis, viability and quantitative culture results approximately halved (both 0.27 log reduction, P <.001) daily. For patients with non-MDR tuberculosis and available data, by treatment day 9 there was a >10-fold reduction in viability in 100% (24/24) of cases and quantitative culture in 95% (19/20) of cases. Four other patients subsequently found to have MDR tuberculosis had no significant changes in viability (P =.4) or quantitative culture (P =.6) results during early treatment. The change in viability and quantitative culture results during early treatment differed significantly between patients with non-MDR tuberculosis and those with MDR tuberculosis (both P <.001). Acid-fast microscopy results changed little during early treatment, and this change was similar for non-MDR tuberculosis vs MDR tuberculosis (P =.6). Conclusions. Tuberculosis quantitative viability microscopy is a simple test that within 1 hour predicted quantitative culture results that became available weeks later, rapidly indicating whether patients were responding to tuberculosis therapy.