One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a simple yet universal high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method to separate molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid profiling using full scan mass spectrometry, as well as lipid class specific scanning in positive and negative ionisation mode. This allows high-throughput processing of samples for lipidomics, even if different types of MS analysis are required. Excellent separation of isobaric and even isomeric species is achieved, and original levels of lyso-lipids can be determined without interference from lyso-lipids formed from diacyl species by source fragmentation. As examples of application of this method, more than 400 phospholipid species were identified and quantified in crude phospholipid extracts from rat liver and the parasitic helminth Schistosoma mansoni. Copyright (C) 2008 John Wiley & Sons, Ltd.