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Objective Of the highly accurate tests for current Helicobacter pylori infection, the urea breath test (UBT) and stool antigen test (SAT) are noninvasive and do not require endoscopy. We conducted a prospective study to evaluate the accuracy of the newly developed SAT in a medical checkup setting. Methods The accuracy of the proposed SAT was examined by determining H. pylori infection status based on a history of eradication therapy, endoscopic H. pylori infection diagnosis, and blood tests (serum H. pylori antibody, serum PG II) in individuals undergoing esophagogastroduodenoscopy (EGD) during a health checkup. Results The new SAT showed 97.3% (108/111) sensitivity for those "currently infected," as well as 99.3% (530/534), 98.0% (402/410), and 98.7% (932/944) specificity for those "never infected," those "previously infected," and those "never/previously infected", respectively. Conclusion The newly developed H. pylori SAT may be useful for diagnosing H. pylori infection. Patients should be suspected of being infected even after H. pylori eradication if they have a high cutoff index in this test.