Tuberculosis is a leading cause of morbidity and mortality worldwide. Susceptibility testing of the causative agent, Mycobacterium tuberculosis, is critical for control of the disease. This study compared the flow cytometric susceptibility assay with the proportion method and the BACTEC TB-460 system. There was agreement between the flow cytometric and proportion methods for 73 (94%) of 78 isoniazid tests, and complete agreement for 26 ethambutol and rifampicin tests. In contrast, the proportion and BACTEC methods failed to agree for 22%, 15% and 8% of isoniazid, ethambutol and rifampicin tests, respectively. These findings indicated that susceptibility testing by the flow cytometric assay is accurate, with results available within 24 h of initiation of the testing procedure.