Lactose-[15N, 15N]-ureide is used to study the fate of the colonic urea-nitrogen metabolism. During the passage through the gastrointestinal tract, lactose ureide is hydrolysed to glucose ureide, which is absorbed to a limited extent from the small intestine and is excreted urinarily. In the present study, a procedure has been developed to quantify the urinary excretion of glucose-[15N, 15N]-ureide. In addition, urine and faecal samples obtained during a dietary intervention study with the prebiotic lactulose were retrospectively analysed. The glucose ureide and lactose ureide content was measured by GC–MS in 19 healthy volunteers. After consumption of a standard test meal containing 75 mg lactose-[15N, 15N]-ureide, six healthy volunteers performed a fractionated 24 h urine collection to investigate the urinary excretion of glucose-[15N, 15N]-ureide. In 13 volunteers, the effect of lactulose administration on the urinary excretion of glucose-[15N, 15N]-ureide was analysed. The urinary excretion of glucose-[15N, 15N]-ureide reached its maximum level in the 3–6 h urine collection and decreased in the 6–9 h urine. The label was still detectable in the 9–24 h urine collection. The cumulative excretion of 15N-labelled glucose ureide after 24 h amounted 12.91%. No significant differences in glucose-[15N, 15N]-ureide excretion were found in either of the urine fractions after administration of lactulose, compared with baseline. In none of the urine samples lactose-[15N, 15N]-ureide was detected. In conclusion, the results obtained in the present study indicated that the percentage dose glucose-[15N, 15N]-ureide recovered in urine is rather constant and not influenced by the presence of lactulose.