Chloroform, the predominant constituent of solvents used for lipid extraction and chromatography, is believed to give rise to birth defects and serious damage to health, and may also be carcinogenic. Therefore, simple and successful methods have been developed to replace chloroform throughout the isolation of glycosphingolipids (GSLs) by less harmful solvents. Gangliosides of sheep brain (ganglio-series gangliosides GM1, GDla, GD1b and GT1b) and of lymphocyte-derived mouse hybridoma cells (namely GM3) were extracted with six different solvent mixtures. Chloroform:methanol:water (40:80:30, v/v/v) was employed as reference (solvent I). Combinations without chloroform were: n-propanol:water (40:10, v/v) (II), methylisobutylketone:methanol:water (40:80:30, v/v/v) (III), ethylacetate:methanol:water (40:72:28, v/v/v) (IV), methylacetate:methanol:water (40:72:28, v/v/v) (V) and petroleum ether:isopropanol:water (40:112:38, v/v/v) (VI). After extraction and dialysis, the weight of lipid extract as well as the content of sialic acid, gangliosides, sulphatides and phospholipids were determined. Quantitation of GSL yields in crude extracts obtained by the alternative solvent mixtures II to VI showed recoveries of brain gangliosides from nearly 67% up to 104% compared with the reference solvent I. Extraction of hybridoma cells by means of the alternative combinations without chloroform revealed at least the same and mostly better ganglioside yields in the range from 98% to 116% with regard to the reference solvent I. n-Propanol:water (II) and methylisobutylketone:methanol:water (III) were the recommended extractants for both tissues. Therefore, the methods described offer simple, less hazardous and successful strategies for GSL extraction in excellent yield without the need for using chloroform.