Abstract: With the object of providing a method for separating glycolipids (particularly gangliosides) capable of processing a large number of samples easily and at low cost, and capable of recovering multiple types of glycolipids with high recovery, in the method for separating glycolipids of the present invention (a) a sample solution obtained by hydrolysis of an extract derived from a biological sample with a mixture of a nonpolar solvent and a polar solvent is first brought into contact via a semipermeable membrane with a solution having lower osmotic pressure than the sample solution, and (b) the contact is continued until the sample solution divides into two or three layers, and the middle layer and/or bottom layer are/is separated.