The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high-abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high-abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow™ technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2-DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers.