This prospective study evaluated the performance of the microscopic observation drug susceptibility (MODS) assay for the direct detection of Mycobacterium tuberculosis drug resistance. MODS assay sensitivity, specificity, and positive and negative predictive values were 96.7% (95% confidence interval [95% CI], 92.1 to 98.8%), 78.4% (95% CI, 73.5 to 80.6%), 82.4% (95% CI, 78.4 to 84.2%), and 95.8% (95% CI, 89.9 to 98.5%), respectively, for isoniazid resistance and 96.0% (95% CI, 90.3 to 98.6%), 82.9% (95% CI, 78.8 to 84.7%), 80.0% (95% CI, 75.2 to 82.1%), and 96.7% (95% CI, 91.9 to 98.8%), respectively, for rifampin resistance. For both rifampin and isoniazid testing, the likelihood ratio for a negative test was < or =0.05, indicating that the MODS assay may be useful for ruling out drug resistance.