Protein/Peptide fractionation plays a critical role in delving deeper into the proteome and is a dynamic area that continually is improving. Various HPLC approaches such as reverse phase HPLC, chromatofocusing, 2D LC, strong cation exchange and avidin chromatography are in use in the Keck MS & Proteomics Resource. Immunoaffinity partitioning is used to deplete biological fluids (serum/plasma, urine, and CSF) of the high and moderate abundant proteins. Finally, gel electrophoresis remains a very powerful protein and peptide fractionation step, with > 2000 proteins separated in a single 2D gel. Newer fractionation approaches using protein isoelectric points are the Agilent OFFGEL 3100 Fractionator and the Protein Forest dPCTM Fractionator, Model SB-106.