We report a carbohydrate microarray-based approach for the rapid, facile analysis of glycosaminoglycan-protein interactions. The key structural determinants responsible for protein binding, such as sulfate groups that participate in the interactions, were elucidated. Specificities were also readily compared across protein families or functional classes, and comparisons among glycosaminoglycan subclasses provided a more comprehensive understanding of protein specificity. To validate the approach, we showed that fibroblast growth factor family members have distinct sulfation preferences. We also demonstrated that heparan sulfate and chondroitin sulfate interact in a sulfation-dependent manner with various axon guidance proteins, including slit2, netrin1, ephrinA1, ephrinA5, and semaphorin5B. We anticipate that these microarrays will accelerate the discovery of glycosaminoglycan-binding proteins and provide a deeper understanding of their roles in regulating diverse biological processes.