Background: Simple, reproducible assays are needed for the quantification of sphingolipids, ceramide (Cer), and sphingoid bases. We developed an HPLC method for simultaneous quantification of total plasma concentrations of Cer, glucosylceramide (G1cCer), and ceramide trihexoside (CTH). Methods: After addition of sphinganine as internal calibrator, we extracted lipids from 50 [micro]L plasma. We deacylated Cer and glycosphingolipids by use of microwave-assisted hydrolysis in methanolic NaOH, followed by derivatization of the liberated amino-group with o-phthaldialdehyde. We separated the derivatized sphingoid bases and lysoglycosphingolipids by HPLC on a C18 reversed-phase column with a methanol/water mobile phase (88:12, vol/vol) and quantified them by use of a fluorescence detector at [[lambda].sub.ex] 340 nm and [lambda].sub.em] 435 nm. Results: Optimal conditions in the Solids/Moisture System SAM-155 microwave oven (CEM Corp.) for the complete deacylation of Cer and neutral glycosphingolipids without decomposition were 60 min at 85% power, fan setting 7. Intra- and interassay CVs were Conclusions: HPLC enables quantification of total Cer, G1cCer, and CTH in plasma and is useful for the follow-up of patients on therapy for Gaucher or Fabry disease.