An improved analytical strategy for the analysis of complex lipid mixtures using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with high-performance thin-layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and -sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post-source decay (PSD) product ion analysis was performed in order to obtain further structural information (head- and acyl-group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R-250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI-MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on-line HPTLC/MALDI-MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI-MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 microL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF-MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples. Copyright (c) 2009 John Wiley & Sons, Ltd.