Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of alpha-galactosidase A, resulting in accumulation of the principal substrate, globotriaosylceramide (Gb(3)), in various physiological fluids and tissues in affected patients. The recognition that accumulation of Gb(3) begins in utero, combined with the fact that the diagnosis of the disease is often delayed until after the development of irreversible tissue damage, has generated pressure to develop techniques for the early, pre-symptomatic diagnosis of the disease. Measurements of urinary Gb(3) have been shown to be useful for the diagnosis of Fabry disease in adults. The objective of this work was to measure the Gb(3)/creatinine biomarker in urine of healthy infants from birth to 6 months, including the establishment of reference ranges for urinary Gb(3) excretion at various postnatal ages, in male and female infants. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine Gb(3)/creatinine ratios in urine specimens dried on filter paper and mailed to the laboratory by participating parents. A total of 728 urine specimens were obtained at intervals from birth to 6 months of age from 68 healthy infants (35 male and 33 female). Parental participation was good, with 90% of the expected specimens received by the laboratory. The results of the analyses were grouped by the age of the infants into four periods. We have determined that both postnatal age and sex have an effect on urinary Gb(3) excretion levels which vary considerably in newborns. We conclude that screening for Fabry disease by measurement of urinary Gb(3) excretion is unlikely to be reliable before 30 days of age.