A method for the analysis of pure samples of individual glycosphingolipids by microscale methanolysis, peracetylation, and gas chromatography is described. Solvolysis of glycosphingolipids in dry methanolic HCl and peracetylation were conducted in a single 4.5-cm sealed capillary tube (2 mm i.d.), after which the products were directly injected into a gas chromatograph. Total-component analysis (i.e., analysis of the sugar, fatty acid, and sphingosine moieties) was possible after a 45-min chromatographic run. Time-course studies of the acid-catalyzed methanolysis of Galβ1-4GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1Cer ganglioside at 80, 110, and 150°C showed that methanolysis was complete after 2 h at 110°C. Rates of methanolysis of individual components were compared and the release of the fatty acid moiety from the long-chain base was shown to be the slowest reaction. The methanolysis of all glycosidic bonds was complete in 0.5 h. Peracetylated methanolysis products were very stable over time and provided for good gas chromatographic detection of subnanomolar amounts of hexose, hexosamine, fatty acid, sialic acid, and long-chain sphingoid base components. Recoveries of fucose and N-acetylglucosamine were determined with reference samples of Fucα1-2Lac and lacto-N-fucosylpentaose II. Applications of the method are presented for the component analysis of a gift mixture of NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-1Cer ganglioside and NeuAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-Cer ganglioside and analysis of NeuAcα2-3Galβ1-4Glcβ1-1Cer isolated from human plasma.