Introduction: WHO has rolled out rapid molecular methods for detection of rifampicin resistance. However, the same are not yet accessible at all the laboratories which are still dependent on alternative methods. Direct colorimetric dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide tube assay (DMTT) for rifampicin susceptibility on sputum, has been shown to be fairly rapid, sensitive and specific but its evaluation has not been reported based on volume of sputum or Acid Fast Bacilli (AFB) grading which is still the routine method for quantifying the bacilli on smear microscopy. Aim: To standardise the DMTT test against volume, grading and Turnaround Time (TAT), and to evaluate the results on clinical samples. Materials and Methods: The present cross-sectional study, duly approved by Institutional Ethics Committee and with written consent from all the patients, was conducted on the sputum samples received in the Department of Microbiology of the Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India, between January 2010 to September 2011. Taking Indirect Agar Proportion Method (IAPM) as reference standard, the test was first standardised with 36 sputum samples of different grades of AFB positivity. The samples were concentrated by Modified Petroff’s method and directly processed for DMTT rifampicin susceptibility test as per protocol. Subsequently 153 clinical samples were similarly evaluated. Results: The test was sensitive even for scanty positive sputum and all results were available by 21st day. For 3+ grade 96% results were ready by 14th day. Increase in volume of sputum decreased the TAT in all grades. DMTT showed good concordance with IAPM in all grades of sputum (91.4% to 100%), overall 95.48%, and could be easily read by naked eye. Conclusion: The DMTT rifampicin test can be performed in all grades of positive sputum and the TAT in scanty and low positive samples can be enhanced by increasing the volume to at least three mL.