Significance Although heparan sulfate (HS) modified by a 3- O -sulfate (3-OS) has been implicated in many biological processes, there is only a rudimentary understanding of ligand requirements of HS-binding proteins requiring this type of modification. We developed a modular synthetic approach that provided a structurally diverse collection of HS oligosaccharides with and without 3-OS. The compounds were printed as a glycan microarray to determine ligand requirements of HS-binding proteins, which revealed specific HS sequons for binding, and led to the discovery of proteins needing 3-OS for binding. The compounds made it possible to determine receptor requirements of herpes simplex virus, and substrate specificities of heparin lyases. The platform provides an important means to examine the biology of 3-OS−modified HS.