To validate a simplified 13C-urea breath test (13C-UBT) method for the diagnosis of H. pylori infection. Patients referred for gastrointestinal endoscopy and biopsy were included, and a 13C-UBT was performed after a 6-hour fast. Breath samples were collected in 10 ml glass tubes before and 30 min after the simultaneous administration of 50 mg of 13C-urea and 2 g of citric acid in 200 ml of water. All breath samples were analyzed using isotope ratio mass spectrometry. The diagnosis of H. pylori infection was established with a positive culture and/or positive histology and serology. Eighty-eight patients were included, 49 female and 39 male with a mean age of 45 +/- 15 yrs. Fifty-one patients (57.95%) were positive and 30 (34.1%) negative for H. pylori. Seven cases (7.95%) were considered undetermined. The sensitivity, specificity, positive predictive value, and negative predictive value for 13C-UBT were 90.2, 93.3, 95.83, and 84.8%, respectively. Accuracy was 91.4%. The simultaneous administration of 50 mg of 13C-urea and 2 g of citric acid represents an alternative for the non-invasive diagnosis of H. pylori infection.