Historically, therapeutic monitoring for prescribed eradication treatment of Helicobacter in cheetahs (Acinonyx jubatus) with associated gastritis has been accomplished only through endoscopic biopsies. The 13C-urea breath test (UBT) can offer an alternative to repeated biopsies for therapeutic monitoring. Five male and five female cheetahs and one male Sumatran tiger (Panthera tigris) were studied. All were clinically healthy before and after this investigation. Breath samples of end-tidal expiration were taken before and after administration of a 13C-enriched urea solution through a gastroesophageal tube. Twenty-milliliter breath samples were taken at 10, 20, 30, and 40 min after administration of the urea solution. The results of the breath analysis were compared with the results of rapid urease testing, histopathologic examination, and impression smears of gastric biopsies taken at the time of the breath test. The sensitivity and specificity for the 13C-UBT in this investigation were 100%. and the positive predictive value and negative predictive value were both 100%. Although the 13C-UBT is a good noninvasive diagnostic tool for monitoring the presence of Helicobacter sp. in the gastric mucosa, endoscopy should still be used for initial diagnosis and grading of gastritis and for monitoring the progression of disease in cheetahs. The 13C-UBT is a valuable, simple, accurate, and sensitive tool for monitoring eradication of Helicobacter during therapy for clinical gastritis.