PURPOSE: To develop a method for the combined analysis of plasma and serum glucosylsphingosine (lyso-Gb1) and globotriaosylsphingosine (lyso-Gb3), biomarkers of Gaucher disease (GD) and Fabry disease (FD), respectively. METHODS: Internal standards were added to 100 µL of plasma/serum and glycosphingolipids: lyso-Gb1, lyso-Gb3, and galactosylsphingosine (GalSph) were extracted with dichloromethane/methanol and analyzed by UPLC-MS/MS. Samples from unaffected controls and patients with GD were first analyzed using a HILIC column to separate lyso-Gb1 from its isomer, GalSph. Samples from patients with FD or GD were analyzed using a C18 column to measure lyso-Gb3 and the hexosylsphingosine (HexSph: lyso-Gb1 + GalSph) fraction in a single combined method. RESULTS: Extraction efficiency was between 73% and 87% and day-to-day variability showed a relative standard error of <7.5%. GalSph was determined to have minimal to no contribution to the HexSph fraction in samples from unaffected controls and patients with GD. Lyso-Gb3 and HexSph measurements by the combined method were in good agreement with established methods, with no bias. CONCLUSIONS: HexSph and lyso-Gb3 analysis by reversed-phase chromatography UPLC-MS/MS is a cost-effective, time-efficient approach for evaluating these glycosphingolipid biomarkers in patients with a suspected or confirmed diagnosis of GD and FD.