Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme α-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb₃), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb₃ in urine samples collected on filter paper, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb₃ techniques being both time- and labour-intensive, this filter paper method eliminates lipid extraction, glycolipid isolation, centrifugation and evaporation steps, while maintaining sensitivity and efficiency. The stability of Gb₃ on filter paper was good for a 7-week period under different temperature conditions. Normal control values were established and the technique was tested with anonymized samples from Fabry hemizygotes and heterozygotes. The levels of total Gb₃ in all classical hemizygotes were well above the control values and all heterozygotes, except two nonexcretors, were above the reference level. The proposed novel filter paper method favours the collection, storage and shipment of samples. It is simple and efficient for a feasibility study, potentially applicable to the determination of total urinary Gb₃ in the newborn population as part of a screening programme, and could also be used in high-risk screening laboratories. Since the incidence of Fabry disease is hard to establish, owing to the heterogeneous clinical expression of the visible phenotype, this feasibility study could help determine its actual incidence in the Quebec population.