The most appropriate time for screening for Fabry disease (FD) is school age. For this reason, we developed non-invasive methods for measuring urinary alpha-galactosidase A (alpha-gal A) protein, using enzyme-linked immunosorbent assay (ELISA), and for globotriaosylceramide (GL-3), using tandem mass spectrometry (MS/MS). We measured these two biomarkers in the urine of previously diagnosed FD hemizygotes and heterozygotes, and in controls. All the classic FD hemizygotes were clearly distinguished from controls by either method alone, and combining the two assays produced 96% sensitivity for detecting heterozygotes. To assess the utility of these methods for screening school children and adults at high risk of FD, a pilot study was conducted. To distinguish FD from 432 controls, cut-off values for alpha-gal A protein and GL-3 were set at the 5th and 95th centile values of the controls, respectively. Among the high-risk patients, the measurements exceeded the cut-off values for both biomarkers in male and female subjects and were strong indicators for Fabry hemizygotes and heterozygotes. However, we recommend that if the results of the first measurements exceed the cut-off values for only one of these biomarkers, another urine sample should be requested for re-assay to confirm the result.