The utility of a line probe assay (Genotype MTBDRplus) performed directly on 2-month sputa to monitor tuberculosis treatment response is unknown. We assessed if direct testing of 2-month sputa with MTBDRplus can predict 2-month culture conversion and long-term treatment outcome. Xpert MTB/RIF-confirmed rifampin-susceptible tuberculosis cases were recruited at tuberculosis diagnosis and followed up at 2 and 5 to 6 months. MTBDRplus was performed directly on 2-month sputa and on all positive cultured isolates at 2 and 5 to 6 months. We also investigated the association of a positive direct MTBDRplus at 2 months with subsequent unsuccessful tuberculosis treatment outcome (failure/death during treatment or subsequent disease recurrence). A total of 279 patients (62% of whom were HIV-1 coinfected) were recruited. Direct MTBDRplus at 2 months had a sensitivity of 78% (95% confidence interval [CI], 65 to 87) and specificity of 80% (95% CI, 74 to 84) to predict culture positivity at 2 months with a high negative predictive value of 93% (95% CI, 89 to 96). Inconclusive genotypic susceptibility results for both rifampin and isoniazid were seen in 26% of MTBDRplus tests performed directly on sputum. Compared to a reference of MTBDRplus performed on positive cultures, the false-positive resistance rate for direct testing of MTBDRplus on sputa was 4% for rifampin and 2% for isoniazid. While a positive 2-month smear was not significantly associated with an unsuccessful treatment outcome (adjusted odds ratio [aOR], 2.69; 95% CI, 0.88 to 8.21), a positive direct MTBDRplus at 2 months was associated with an unsuccessful outcome (aOR 2.87; 95% CI, 1.11 to 7.42). There is moderate utility of direct 2-month MTBDRplus to predict culture conversion at 2 months and also to predict an unfavorable outcome.