Heparan sulfate is a sulfated polysaccharide that displays essential physiological functions. Here, we report a LC-MS/MS-based method for quantitatively determining the individual disaccharide composition and total amount of heparan sulfate. Using eight 13C-labeled disaccharide calibrants and one 13C-labeled polysaccharide calibrant, we complete the analysis in one-pot process. The method is both sensitive and has the throughput to analyze heparan sulfate from mouse tissues and plasma.