Purpose Anti-tuberculosis drug susceptibility testing (DST) is increasing in importance and demand for treatment. Supply of standardized high-quality DST medium and tuberculosis (TB) specimen titration are key technologies in the DST process. The purpose of this study was to evaluate test method that secures accuracy by taking advantage of the proportion tests. Methods To perform the anti-TB DST, an improved 6-well medium (Middlebrook 7H10 medium) produced in a GMP-qualified place was used. After inoculation in 6-well medium, it was compared with the result of REMA. The concentration of the drug added for the drug susceptibility test was based on the value recommended by WHO. Results The improved 6-well anti-TB DST medium produced in a GMP certified facility is expected to be able to replace the in-house medium used for phenotypic DST. Conclusions It is thought that the medium used for phenotype DST should be produced in a GMP certified facility in order to standardize and high quality. The improved 6-well medium is a meaningful attempt to improve the quality of DST. For the clinical introduction of an improved medium, additional experiments and verification work are required for TB strains with various drug resistance patterns.