Heparins are negatively charged polydispersed linear polysaccharides which have the ability to bind a wide range of biomolecules including enzymes, serine protease inhibitors, growth factors, extracellular matrix proteins, DNA modification enzymes and hormone receptors. In this chromatography, heparin is not only an affinity ligand but also an ion exchanger with high charge density and distribution. Heparin chromatography is an adsorption chromatography in which biomolecules can be specifically and reversibly adsorbed by heparins immobilized on an insoluble support. An advantage of this chromatography is that heparin-binding proteins can be conveniently enriched using its concentration effect. This is especially important for separating low abundance proteins for the analysis in two-dimensional electrophoresis (2DE) or other proteomics approaches. Heparin chromatography is a powerful sample-pretreatment technology that has been widely used to fractionate proteins from extracts of prokaryotic organism or eukaryotic cells. As an example, the fractionation of fibroblast growth factors (FGFs) from the extract of mouse brain microvascular endothelial cells (MVEC) is now introduced to demonstrate the procedure of heparin chromatography.