Background: Tuberculosis (TB) still a major health problem worldwide. Emerging Multidrug-Resistant Tuberculosis-TB (MDR-TB) is an important problem facing the health policy. Actually, less than 10% of MDR-TB cases worldwide are detected. The conventional microscopy ZN (Ziehl-Neelsen) technique lacks sensitivity. The cultural methods which have high sensitive takes as long as 14 days up to 6 weeks to produce results. The Xpert assay is a promising approach for the rapid diagnosis of active TB which provides results within 2 hours. Objectives are to compare the sensitivity and specificity of Gene Xpert/RIF assay versus the traditional methods in detection of Mycobacterium tuberculosis and evaluation of rifampicin resistance in sputum samples. Methodology: The study was carried out on 30 cases under clinical and radiological doubt of pulmonary tuberculosis from May 2017 to January 2018.three consecutive spontaneously produced early morning sputum samples were collected and subjected to ZN smear microscopy, culture and conventional drug susceptibility test (DST) followed by Gene Xpert MTB/RIF assay. The sensitivity, specificity, PPV, NPV of Gene Xpert & ZN microscopy were calculated using LJ culture of Mycobacterium tuberculosis as standard method Results: A total of 30 sputum samples were tested; among the 30 sputum samples, 8 samples were positive and 18 samples were negative by the three methods used (smear microscopy, culture, gene xpert assay).the overall sensitivity, specificity, PPV & NPV of gene Xpert assay were 100%, 94.7%, 91.7% and100% respectively. The overall sensitivity, specificity, PPV & NPV of smear microscopy were 72.7%, 100%, 100% and 86.3% respectively. Sensitivity, specificity, PPV & NPV of Gene Xpert for detecting RIF resistance compared to phenotypic DST were 100%,100%,100% and100% respectively. Conclusion: GeneXpert MTB/RIF assay is simple, efficient and accurate technique for the rapid diagnosis of MTB. It’s simplicity, high sensitivity and specificity for detection of RIF resistance make this technique a very impressive tool for diagnosis of MTB and RIF resistance in MDR cases.