Rapid and inexpensive assays for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are urgently required especially in developing countries where tuberculosis cases are prevalent. In response to this necessity, a direct microplate-based colorimetric assay which excludes the use of pre-testing culture isolate was evaluated. MTB susceptibility to the first line anti-tuberculosis drugs was tested directly on sputum specimens using tetrazolium microplate assay (TEMA) method and the sensitivity, specificity, accuracy as well as mean turn-around time of TEMA were compared to the standard absolute concentration method (ACM). TEMA was performed on 41 acid fast bacilli (AFB) positive sputum specimens by direct inoculation of the processed specimens into the microplate wells containing serialdiluted first line anti-tuberculosis drugs using tetrazolium dye as growth indicator. Indirect TEMA was performed on MTB isolates of the corresponding samples. The minimum inhibitory concentrations (MICs) of isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SM) were obtained for direct and indirect TEMA with reference to the absolute concentration method (ACM). After establishing the breakpoint MIC of each drug using receiver operating characteristics (ROC) curve, reliable results from direct TEMA were obtained for INH and SM, with excellent levels of sensitivity, specificity, and accuracy (more than 90%). The predictive values for susceptibility were 100% for INH, EMB and SM as well as 96% for RMP. A shorter mean turn-around time of 14 days was observed for direct TEMA (P < 0.05). Thus direct TEMA is potentially rapid, reliable and inexpensive DST screening method of MTB in countries with high prevalence rates of drug resistance tuberculosis