Elucidation of the relationship between the structure and biological function of the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS) presents an important analytical challenge mainly due to the difficulty in determining their fine structure. Heparin and HS are responsible for mediation of a wide range of biological actions through specific binding to a variety of proteins including those involved in blood coagulation, cell proliferation, differentiation and adhesion, and host–pathogen interactions. Therefore, there is a growing interest in characterizing the microstructure of heparin and HS and in elucidating the molecular level details of their interaction with peptides and proteins. This review discusses recent developments in the analytical methods used for sensitive separation, detection, and structural characterization of heparin and HS. A brief discussion of the analysis of contaminants in pharmaceutical heparin is also presented.