PURPOSE: A simple, rapid, and highly sensitive analytical method for Gb3 in plasma was developed without labor-extensive pre-treatment by electrospray ionization MS/MS (ESI-MS/MS). Measurement of globotriaosylceramide (Gb3, ceramide trihexoside) in plasma has clinical importance for monitoring after enzyme replacement therapy in Fabry disease patients. The disease is an X-linked lipid storage disorder that results from a deficiency of the enzyme ??-galactosidase A (alpha-Gal A). The lack of alpha-Gal A causes an intracellular accumulation of glycosphingolipids, mainly Gb3. METHODS: Only simple 50-fold dilution of plasma is necessary for the extraction and isolation of Gb3 in plasma. Gb3 in diluted plasma was dissolved in dioxane containing C17:0 Gb3 as an internal standard. After centrifugation it was directly injected and analyzed through guard column by in combination with multiple reaction monitoring mode of ESI-MS/MS. RESULTS: Eight isoforms of Gb3 were completely resolved from plasma matrix. C16:0 Gb3 occupied 50% of total Gb3 as a major component in plasma. Linear relationship for Gb3 isoforms was found in the range of 0.001-1.0 microgram/mL. The limit of detection (S/N=3) was 0.001 microgram/mL and limit of quantification was 0.01 microgram/mL for C16:0 Gb3 with acceptable precision and accuracy. Correlation coefficient of calibration curves for 8 Gb3 isoforms ranged from 0.9678 to 0.9982. CONCLUSION: This quantitative method developed could be useful for rapid and sensitive 1st line Fabry disease screening, monitoring and/or diagnostic tool for Fabry disease.