A method is described for the quantitative isolation and estimation of the four glycosphingolipid fractions from 10 ml of human plasma. The procedure consists of acetylation of the total lipids after extraction, separation of acetylated glycophingolipids from non-glycolipids on a Florisil column, deacetylation, dialysis in water, separation of the single fractions by thin-layer chromatography on silica gel, and quantitative analysis of each glycosphingolipid by sulfuric acid-orcinol reagent, carried out in the presence of silica gel. The concentrations of the four glycosphingolipids in human plasma derived from a group of 23 healthy subjects are presented. The procedure described is sufficiently sensitive for clinical investigation.