The aim of this study was to evaluate the utility of the quantitative analysis of mycolic acids by HPLC technique in drug susceptibility testing of the M. tuberculosis isolates to the first-line antituberculous drugs: isoniazid and rifampicin. Drug susceptibility of the 30 clinical M.tbc isolates was examined by the mycolic acids analysis with HPLC technique and results were compared to the proportion method on solid L-J medium and liquid medium in MGIT system. In HPLC method drug susceptibility of M. tuberculosis strains was described by TAMA index defined as the ratio of the total area under mycolic acids peaks (TAMA) from cultures with drug to the TAMA of control. At critical concentrations of drugs, TAMA indexes of resistant strains were >0.5, and TAMA indexes of susceptible strains were <0.05. The average error of the TAMA analysis was +/- 9.5% The quantitative analysis of mycolic acids by HPLC gives results compatible with standard proportion method and is a reliable method for determination of drug susceptibility of M. tuberculosis.