A method is described which permits the quantitative microanalysis of glycosphingolipids in the picomole range by HPLC of their perbenzoylated derivatives with detection at 230 nm. A linear gradient of 1 to 20% dioxane in hexane is used to elute from a Zipax column derivatives that contain up to four sugar residues. Residual absorption due to dioxane is negated by directing the solvent gradient through a pre-column, pre-injector high-pressure reference cell. This path generates a horizontal baseline with a negative and a positive deflection at the beginning and end of the gradient, respectively. Neutral glycosphingolipids can be quantitated either with N-acetyl-psychosine as internal standard or by comparison of peak areas to those of external standards.