A method is described for the determination of plasma and serum glycosaminoglycans, which can be used in any laboratory equipped with an HPLC system. It is based on the sequential application of chondroitinases AC and ABC and separation of the resulting disaccharides by high-performance liquid chromatography. All reagents are commercially available. This simple and rapid separation yields an accurate quantification and an exact distribution pattern. The determination of glycosaminoglycan disaccharides is linear between 7 and 7000 mumol/l with coefficients of variation between 3.0 and 7.7% for serum and between 2 and 14% for plasma. The recovery of the assay ranged from 93 to 106% for different concentrations of glycosaminoglycan disaccharides. This HPLC method may therefore be considered as a candidate reference method.