Changes in sphingolipids have been associated with profound effects in cell fate and development in both plants and animals. Sphingolipids as a group consist of a large number of different compound classes of which numerous individual species may vary in response to environmental stimuli to affect cellular responses. The ability to measure all sphingolipids simultaneously is, therefore, essential to an understanding of the biochemical regulation of sphingolipid metabolism and signaling molecules derived from it. In the model plant Arabidopsis thaliana, the major sphingolipid classes are glycosylinositolphosphoceramides, glucosylceramides, hydroxyceramides and ceramides. Other minor but potentially important sphingolipids are free long-chain bases and their phosphorylated derivates. By using a single solvent system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry detection we have been able to separate and measure 168 sphingolipids from a crude sample. This greatly speeds up and simplifies the analysis of plant sphingolipids and should pave the way for a better understanding of their role in plant performance. Copyright © 2007 John Wiley & Sons, Ltd.