Heparan sulfate 6-endosulfatases (SULFs) remove 6-O-sulfo groups from heparan sulfate polysaccharide chains. SULFs modify the functions of heparan sulfate and contribute to the development of cancers, organ development and endothelial inflammatory responses. However, direct measurement of the activity of SULFs from human and mouse plasma is not currently possible. Here, we report a liquid chromatography coupled with tandem mass spectrometry (LS-MS/MS) assay to measure the activity of SULFs. The method uses a structurally homogeneous heparan sulfate dodecasaccharide (12-mer) in which the glucuronic and iduronic acid residues are labeled with both 13C- and 2H-atoms. The 12-mers desulfated by the SULFs is subjected to degradation with heparin lyases to yield disaccharides, which is followed by LC-MS/MS. The amount of two specific disaccharides, ΔIIIS and ΔIVS, quantified by LC-MS/MS reports the activity of the SULFs with high sensitivity and specificity. This method allows for the determination of the activity from conditioned cell media and mouse plasma. Our findings offer an essential novel tool to delineate many roles of SULFs in biological processes.