Most of the introduced susceptibility methods of Mycobacterium tuberculosis have some disadvantages for screening. Therefore, the selection of susceptibility assay for evaluating candidate agents must be determined case by case. In this study, we evaluated the validity of a modified broth dilution-based assay in comparison to the gold standard proportional method for microbial sensitivity test of new lipophilic compounds candidate as antitubercular agents. The in-vitro susceptibilities of 114 M. tuberculosis strains were separately tested against isoniazid and two lipophilic antitubercular agents (derivative of dihydropyridines) by employing the standard proportional method and a modified broth dilution-based assay. The results for isoniazid testing showed 100% concordance for sensitivity, specificity and reproducibility. In the case of microbial sensitivity test of lipophilic compounds, comparison of the results obtained from these two methods indicates a significant superiority of the modified method over the standard method. Considering the other advantages of this modified method, we concluded that this modified broth dilution-based assay could be utilized effectively for the susceptibility testing of new lipophilic compounds candidate as antitubercular agents. © 2016 Published by Elsevier Ltd.