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The interplay between resolution, accuracy, sensitivity and speed of the mass spectrometer, as well as the complexity of the peptide mixture in relation to chromatographic separation and the analysis time finally determines the number of identified proteins within a ‘shotgun’ proteomic study. The improvement of one of these parameters can enhance the quality of the proteome analysis. Here we evaluated the technique of in-solution isoelectric focusing (IEF) for pre-fractionation of tryptic peptides prior to LC-MS/MS-based proteomics analysis. In-solution IEF turned out to be a simple and fast method for peptide fractionation prior to LC-MS analysis. By adapting the experimental procedures, this approach enabled identification of more than 44,000 peptides belonging to 5,800 proteins in less than 48 working hours, from protein extraction until the end of LC-MS analysis. This technique was applied successfully to analyze the proteomes of mammalian cells and different model organisms, without additional efforts or special technical equipment. The in-solution IEF of peptides is very robust and can be applied in combination with different extraction procedures. The high number of identified peptides using a standard LC-MS system led to average protein coverage of 25%. Such a high average number of identified peptides per protein improved the discrimination of protein species as isoforms or splice variants. Thus, in solution IEF is a fast and robust alternative to gel-based proteomics or other gel free fractionation techniques upstream to LC-MS/MS analysis. The reduction of processing time and the high performance of this technique can speed up deep proteomics analyses significantly.