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We have developed a protocol suitable for high-throughput lipidomic analysis of human brain samples. The traditional Folch extraction (using chloroform and glass-glass homogenisation) was compared to a high-throughput method combining methyl-tert-butyl ether (MTBE) extraction with mechanical homogenisation utilising ceramic beads. This high-throughput method significantly reduced sample handling time and increase efficiency compared to glass-glass homogenising. Furthermore, replacing chloroform with MTBE is safer (less carcinogenic/toxic), with lipids dissolving in the upper phase, allowing for easier pipetting and the potential for automation (i.e. robotics). Both methods were applied to the analysis of human occipital cortex. Lipid species (including ceramides, sphingomyelins, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserines) were analysed via electrospray ionisation mass spectrometry and sterol species were analysed using gas chromatography mass spectrometry. No differences in lipid species composition were evident when the lipid extraction protocols were compared, indicating that MTBE extraction with mechanical homogenisation provides an improved method for the lipidomic profiling of human brain tissue.