Recent advances in mass spectrometry have revolutionized the analysis of lipid compositions of cells and other biomaterials by simplifying the analytical protocol dramatically and by increasing the sensitivity of detection by several orders of magnitude. However, the throughput of the published mass spectrometric methods is severely limited by data analysis, which requires extensive operator involvement. Consequently, we have developed an automated method that allows unattended identification and quantification of lipid molecular species of all the major lipid classes from a two-dimensional chromatographic/mass spectrometric data set. More than 100 polar lipid species could be automatically quantified from different biological samples with good accuracy and reproducibility. The response was linear over approximately 3 orders of magnitude with the equipment used, and approximately 35 samples could be analyzed in a day. This method makes high-throughput lipidomics feasible in biology, biotechnology, and medicine.