Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from a deficiency of $alpha$-galactosidase A, which leads to the progressive accumulation of one biomarker, globotriaosylceramide (Gb3), prominently elevated in the urine of affected patients. Using filter paper discs saturated with urine, we evaluated the analytical performance and clinical usefulness of the urinary Gb3 assay by tandem mass spectrometry (LC-MS/MS), with respect to linearity, precision, and reproducibility. We used healthy control urine samples to validate the reference interval of urinary Gb3. This method showed a good linearity ($R^2=0.9998$) in the range of $0.05-10;{mu}g/mL$. Within-run CVs were less than 5% and total CVs were within 10%. The mean recovery of Gb3 from the urine filter paper was 96.7% and the limit of quantification (S/N $geq$ 5) was $0.05;{mu}g/mL$, which was sensitive enough for the diagnosis of FD. The mean concentration of Gb3 in urine samples from healthy Korean controls was $5.93{pm}3.6;{mu}g/mmol$ Cr (range $0.9-16.43;{mu}g/mmol$ Cr). The urinary Gb3 assay by LC-MS/MS showed good analytical performance required for the diagnosis of FD in its linearity, precision, and accuracy. Therefore, this technique could be used for a rapid and reliable first line screening, monitoring and/or diagnosis of individuals at high risk for FD.