Background:Smear negative tuberculosis patients are usually associated with delayed or incorrect diagnosis, as well as poor treatment efficacy. This study aimed to assess the Gene-Xpert MTB/RIF assay performance in detection of Mycobacterium tuberculosis in smear-negative sputum samplesMethods: The study was conducted in Al Quwayiyah General Hospital, Riyadh, Saudi Arabia from January to December 2021 involving 131 patients. Each sputum sample was examined directly by Ziehl-Neelsen stain. All sputum samples were cultured on MGIT tubes using BACTEC MGIT 960 System. GENEXPERT MTB/RIF assay was carried out for collected sputum samples after digestion-decontamination of sputum using MycoPrep. reagent.Results: Out of the 131 studied patients, 24 (18.32%) had positive mycobacterial cultures, of these 24 positive patients, 14 (10.68%) were culture positive smear positive tuberculosis patients, 10 (7.63%) were culture positive smear negative tuberculosis patients and 107 were culture negative patients. Twenty three (17.6%) sputa were positive by both tuberculosis culture and GeneXpert MTB/RIF assay, one sputum sample was culture positive but GeneXpert MTB/RIF assay negative. In comparison to  mycobacterial culture as the gold standard, the sensitivity of the GeneXpert MTB/RIF assay in culture positive/smear positive was 100% (76.84-100%) while the sensitivity of the GeneXpert MTB/RIF technique among culture positive smear negative tuberculosis patients was 90.91% (58.72%-99.77%). The specificity of the GeneXpert MTB/RIF assay was 99.07% (94.90%-99.98%) in culture positive/smear positive patients and it was 98.15% (93.47%-99.77%) among smear culture positive smear negative tuberculosis patients. Conclusion: The Xpert MTB/RIF assay has better sensitivity and specificity which is nearly as same as culture while it is much faster in detection of Mycobacteria.