Introduction: Tuberculosis (TB) presents globally as a significant health problem and health care workers (HCW) are at increased risk of contracting TB infection. There is no diagnostic gold standard for latent TB infection (LTBI). Tuberculin Skin Test (TST) is the traditional testing method for diagnosing LTBI, but it has a known high rate of false positive with subsequent needless loss of time, efforts, loss of productivity and side effects. Interferon-gamma (IFN-γ)-Release Assays (IGRA) has been developed as a potential replacement for the TST. Aim: To assess the prevalence of TB among health care workers in high, middle and low risk areas for TB infection at Cairo University Hospitals (CUH) and to validate the use of Quantiferon as a screening test for TB compared to tuberculin test. Subjects and methods: A cross sectional study with non-random sequential sample of 200 HCWs employed in Cairo University Hospitals and agreed to participate was performed. Information on gender, age, workplace, job title, BCG vaccination and history of both TB and TST were collected using a questionnaire. All participants were subjected to clinical examination, TST, Quantiferon test and chest X-ray. Results: The participants were 70 males and 130 females with mean age + SD (34.7 ± 12.6) years. TST was positive in 86 cases (43%) and the QFT results were positive in 38 cases (19%). There was average agreement between the two tests (76%, к = 0.474), concordance between two tests improved from (к = 0.474) at cut-off point 10 mm to (к = 0.492) at cut-off point 15 mm. LTBI was most common among nurses, workers and bacteriology laboratory technicians in high risk areas, specially in the Chest department, the Bacteriology Lab and the Internal Medicine departments with highest mean diameter of induration for the studied population in the chest department (16.6). According to the nature of work the laboratory technicians had the highest mean diameter of induration (16.58). Thirty-four (17%) of the studied population had abnormal chest radiographs suspected of TB. Occupational exposure in high risk area and working duration >10 years were statistically significant risk factors for both QFT & TST, while age and nature of work were not statistically significant risk factors for both tests using multivariable logistic regression. Conclusion & recommendations: There is risk of tuberculosis transmission among HCWs at Cairo University Hospital, principally among nurses, workers and bacteriology lab technician and in Chest Department, Internal Medicine Department and bacteriology lab. Large scale of screening involving all University hospital staff must be performed. The prevalence of LTBI was 19% by Quantiferon and 43% by TST, this might be due to false-positive TST and that Quantiferon is more specific than TST.Since the Quantiferon is much more expensive than TST therefore we recommend screening of HCWs using TST then confirming those positive TST by Quantiferon and following them yearly using Quantiferon. Disease probability in HCWs tested positive by serial testing should be further assessed by chest radiograph. A treatment regimen for latent TB infection has to be followed and further reinforcement of the infection control policy