Since mammalian glycolipids are minor components except for samples derived from neural tissues, it is necessary to analyze concentrated samples with appropriate methods. Although the method of analyzing concentrated glycolipids by mass spectrometry is already established, further improvements in detection sensitivity and the ability to clarify differences in anomers are required. The components that can be analyzed are limited by the method of concentration, so it is necessary to carefully consider which method to choose. The mass spectrometers used are matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) that can be connected to liquid chromatography, and the choice of either mass spectrometer depends on the purpose of the analysis.