Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum, biomarkers has been impeded by the dominance of human serum albumin. (HSA) and immunoglobulin G (IgG) in the serum, proteome. Therefore, in order to observe lower-abundance serum proteins, removal or depletion of at least these two proteins is required. However, the depletion method needs to be inexpensive and reproducible. We describe such a protocol that combines delipidation by centrifugation, IgG removal with Protein G Sepharose™, and HSA depletion with sodium chloride/ethanol precipitation. The protocol is streamlined to increase reproducibility and is compatible with many proteomie platforms, including two-dimensional gel electrophoresis, and high-performance liquid chromatography either offline or coupled online with a mass spectrometer. The reproducible depletion of lipids, IgG. and HSA permits a higher load of the remaining serum proteins, facilitating the identification of disease biomarkers.