The validity of measuring isotopic enrichment of amino acids in urine was tested in neonates during different feeding regimens and tracer administration protocols, and with different amino acid tracers. Twenty infants, fed enterally or parenterally, were given primed, constant infusions of 15N-glycine, L-[1-13C]leucine, and L-[1-13C]phenylalanine. Urine was collected every 2 h for 16 h. Blood was sampled only when required clinically. At baseline, enrichment in plasma and urine did not differ for any amino acid. At isotopic steady state, leucine enrichment was similar in plasma and urine, while the agreement was slightly weaker for glycine and phenylalanine, probably because of problems inherent in drawing blood samples from neonates. Study protocol did not affect the agreement. Since urine collection involves minimal invasive procedures, this approach could facilitate more indepth tracer studies in infants and children.