Objective To establish of MIC in microtiter as drug susceptibility testing for Mycobacterium tuberculosis(microwell-MIC-test)and evaluate clinical application of this method in Mycobacterium tuberculosis.Methods 60 Mycobacterium tuberculosis clinical isolates which were stored in Shanghai Pulmonary Hospital and whose drug susceptibility status of Bactec MGIT was known were as control.MICs of these 60 Mycobacterium tuberculosis strains were detected in liquid culture in 96-well plate.According to the results of Bactec MGIT.the best cut-off value were chosen by ROC analysis.The drug susceptibility of 80 Mycobacterium tuberculosis clinical isolate strains which were collected continuously from tuberculosis patients in our hospital were detected by Microwell-MIC-test.Bactec MGIT method was used at the sanle time to evaluate and amend the cut-off valne.Results The best concentration of Mycobacterium tuberculosis in microwell is 10~(-3) mg/ml.The best results were read after 7-10 d The best cut-off value of drug susceptibility testing of isoniazid,streptomycin,rifampicin,ethambutol,ofloxacin,amikacin,capreomycin were 2μg/ml,0.25μg/ml,1 μg/ml,2μg/ml,1 μg/ml,4μg/ml and 4μg/ml,respectively,and the seusitivities were 93.5%,97.7%,93.5%,84.4%,95.8%,91.7% and 88.9% respectively,the specificities were 95.6%, 94.6%,100.0%,93.8%,92.9%,95.6% and 91.5% respectively.The accuracies were 95.0%,96.3%,97.5%,90.0%,93.8%,95.0% and 91.3%respectively.Conlusion The drug susceptibility testing method of microwell-MIC-test is accurate,rapid,low-cost and simple and worthy in application in tuberculosis hospital of resource limited countries like China.