Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum early during infection, diagnosis does not usually occur until >3-4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in ten minutes with no processing or technical expertise. This assay utilizes the Mycobacterium tuberculosis-specific biomarker BlaC in reporter enzyme fluorescence (REF) that has been optimized for clinical samples, designated REFtb, along with a more specific fluorogenic substrate, CDG-3. We report the first evaluation of clinical specimens with REFtb assays in comparison to the gold standards for tuberculosis diagnosis, culture and smear microscopy. REFtb assays allowed diagnosis of 160 patients from 16 different countries with a sensitivity of 89% as compared to smear positive culture positive and 88% for smear negative culture positive and a specificity of 82%. The negative predictive value of REFtb for tuberculosis infection is 93% and positive predictive value is 79%. Overall, these data point toward the need for larger accuracy studies by third parties using a commercially available REFtb kit to determine whether incorporation of REFtb into the clinical toolbox for suspected tuberculosis patients would improve case identification. If results similar to our own can be obtained by all diagnostic laboratories, REFtb would allow proper treatment of more than 85% of patients that would be missed during their initial visit to a clinic using current diagnostic strategies, reducing the potential for further spread of disease.