In this study we have investigated whether micro-solution isoelectric focusing (µsol-IEF) can be used as a pre-fractionation step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) and if extensive sample purification of the different fractions is required. We found that, in spite of the high concentrations of buffer and detergents, no clean up of the digested µsol-IEF fractions was necessary before analysis by LC/MS/MS. We also concluded that it is possible to identify at least twice as many proteins in a glioma cell lysate with the combination of µsol-IEF and LC/MS/MS than with LC/MS/MS alone. Furthermore, most of the proteins that were identified from one µsol-IEF fraction by using analytical narrow-range two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were also identified by LC/MS/MS. Finally, we used the combination of µsol-IEF and LC/MS/MS to compare two sample preparation methods for glioma cells and found that several nuclear, mitochondria, and endoplasmic reticulum proteins were only present in the sample that had been subjected to lipid extraction by incubating the homogenized cells in chloroform/methanol/water. Copyright © 2005 John Wiley & Sons, Ltd.