Heparin and pentosan are highly-sulfated glucosaminoglucan, widely used in pharmaceutical industry. Both molecules carry a high negative density attributed to multiple sulfate groups. Due to a very polar and polymeric nature of these compounds, separating and quantifying them is a very difficult task. Heparin and its salt, along with pentosan, are separated on a Primesep N column in HILIC/anion-exclusion mode. Method can be used for quantitation of heparin and pentosan in biofluids and formulations. Elution is monitored by ELSD, CAD or LC/MS.