Objective: Real Time-PCR based Magicplex TB/MDR Test (Seegene, Korea) was evaluated to detect resistance to INH and RIF directly from smear-negative and smear-positive sputum samples. Methods: Sputum samples (N=96) banked in Tuberculosis Specimen BioBank established in Masan National Hospital were randomly selected and applied for smear examination, M. tb isolation by culture, and DNA extraction. Culture based drug susceptibility test (DST) in 7H10 medium as the gold standard of drug resistance was performed with M. tb isolates. DNA samples extracted from both sputum and M. tb isolates were applied for detecting resistance to INH and RIF. Results: Among the sputum samples employed, 42 (43.8%) were smear-negative and 54 were smear-positive with 26 (27.0%) scanty, 12 (12.5%) 1+, 8 (8.3%) 2+, and 8 (8.3%) 3+. Forty-five sputum samples (46.9%) were culture positive. Eight (19%) samples were culture positive among smear negative samples while 41 (76%) of smear positive were positive with 13 (50%) of scanty, 8 (67%) of 1+, 8 (100%) of 2+, and 8 (100%) of 3+. Nine samples were excluded because of culture contamination and NTM isolation. In smear negative samples, sensitivity and specificity of RT-PCR based test for INH were 80.0 and 66.7%, while for RIF, 60.0 and 100%, respectively. In smear positive samples, sensitivity and specificity for INH were 81.3 and 93.3%, and for RIF, 68.2 and 93.3%, respectively. Conclusions: RT-PCR based MagicPlex kit showed an excellent specificity for both INH and RIF among not only smear negative but also smear positive sputum samples.