The proteome of any cell or even any subcellular fraction remains too complex for complete analysis by one dimension of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to achieve greater depth of coverage for a proteome of interest, most groups routinely subfractionate the sample prior to LC-MS/MS so that the material entering LC-MS/MS is less complex than the original sample. Protein and/or peptide fractionation methods that biochemists have used for decades, such as strong cation exchange chromatography (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common prefractionation methods used currently. There has, as yet, been no comprehensive, controlled evaluation of the relative merits of the various methods, although some binary comparisons have been made. Here, we compare the most popular methods for fractionating samples at both the protein and peptide level, replicating all analyses to provide estimates of the variability in the analyses and controlling precisely for instrument time dedicated to each analysis, as well as directly measuring the recovery of protein or peptide from each fractionation procedure. For maximal proteome coverage, SDS-PAGE is very clearly the most effective method tested, with more than 90% of the entire data set found. When considering the amount of material recovered after each fractionation procedure, solution-based IEF and SCX performed similarly, with approximately 80% of the input being recovered.