In this study a simple and effective procedure for the isolation of individual neutral glycosphingolipids (GSLs) by preparative thin-layer chromatography is described. The method is based on nondestructive visualization of neutral GSLs on silica gel precoated thin-layer chromatography plates with anionic lipophilic fluorochromes. After thin-layer chromatography, individual neutral GSLs were detected by spraying the plate with fluorochrome solution followed by exposure to ultraviolet light. GSL containing silica gel was scraped off and extracted with chloroform:methanol:water (30:60:8, by vol). Neutral GSLs were freed from contaminating anionic fluorochrome by DEAE-Sepharose anion-exchange chromatography. Finally, a stepwise chloroform:methanol gradient chromatography on a small silica gel K60 column was employed to remove non-GSL impurities. Of nine different anionic fluorochromes tested, 2-(N-methylanilino)-naphthalene-6-sulfonic acid was found to be the most suitable for preparative purposes. The method was proved with reference GSL mixtures containing glucosyl-, galactosyl-, lactosyl-, globotriaosyl-, and neolactotetraosylceramides, each substituted with C24- and C16-fatty acids, resulting in isolation of individual GSL fractions. The technique was applied for the purification of neutral GSLs from mouse kidney and human granulocytes carrying Lewisx-epitopes. In summary, the method described offers an easy to handle and successful preparative thin-layer chromatography strategy to obtain pure neutral GSLs in microgram and milligram quantities.