Many laboratories validate DST of the second-line drugs by BACTEC MGIT 960 system. The objective of this study is to evaluate the critical concentration and perform DST for the 2nd line drugs. We evaluated 193 clinical strains of M. tuberculosis isolated from patients in South Korea. Testing the critical concentration of six second-line drugs was performed by MGIT 960 and compared with L-J proportion method. The critical concentration was determined to establish the most one that gave the difference between drug resistance and susceptibility in MGIT960 system. Good agreement of the following concentrations was found: Concordance was 95% for 0.5 μ g/mL of moxifloxacin; 93.6%, 1.0 μ g/mL of levofloxacin; 97.5%, 2.5 μ g/mL of kanamycin; 90.6%, 2.5 μ g/mL of capreomycin; 86.2%, 5.0 μ g/mL of ethionamide; and 90.8%, 2.0 μ g/mL of ρ-aminosalicylic acid. The critical concentrations of the four drugs, moxifloxacin, levofloxacin, kanamycin, and capreomycin, were concordant and reliable for testing 2nd line drug resistance. Further study of ethionamide and ρ -aminosalicylic acid is required.