A glycan microarray was developed using standard robotic printing technology to covalently attach amine-functionalized glycans to an amino-reactive glass slide. The array contains 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. This array is highly useful for profiling the specificity of a wide range of glycan-binding proteins (GBPs), including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses. Glycomics, like proteomics, has emerged as an important area of study in the postgenomics era. Glycans are essential for recognition, adherence, motility, and signaling in living systems, and GBPs play a key role in decoding glycan information by recognizing and binding to glycosylated proteins and lipids. However, the complexity of GBP-ligand interactions and the lack of well-defined glycan libraries and efficient screening methods have limited the analysis of GBP specificity and their biological roles. To address these challenges, the development of reliable and efficient tools for analyzing GBP specificity is needed. In recent years, various approaches for constructing glycan arrays for GBP specificity analysis have been developed. These approaches differ in the type of glycans and the method of display. Some use noncovalent attachment to plastic or nitrocellulose membranes, while others use covalent attachment to plastic, gold, or glass. The glycans displayed range from a limited number of exemplary structures to libraries of defined glycans, proteoglycan fragments, and microbial polysaccharides. This study describes a glycan array format that uses standard robotic printing technology to create a diverse glycan array with demonstrated applicability for profiling the specificity of a wide variety of GBPs. The array uses commercially available amine-reactive NHS-activated glass slides, which allow rapid covalent coupling of amine-functionalized glycans or glycoconjugates. The array was developed using a glycan library comprising over 200 synthetic and natural structurally defined terminal sequences of glycoprotein and glycolipid glycans, which could be readily modified to contain amino-functionalized linkers for covalent coupling to NHS-activated glass slides. The array was demonstrated to be useful for analyzing most major classes of GBPs, including mammalian lectins, plant lectins, antibodies, viral and bacterial lectins, and intact viruses. The array was used to analyze the specificity of various GBPs, including plant lectins, human GBPs, and bacterial and viral GBPs. The results showed that the array is highly effective for profiling GBP specificity and revealing unique aspects of fine specificity not previously recognized. The array has potential applications in therapeutic developments and screening for glycan-specific antibodies.A glycan microarray was developed using standard robotic printing technology to covalently attach amine-functionalized glycans to an amino-reactive glass slide. The array contains 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. This array is highly useful for profiling the specificity of a wide range of glycan-binding proteins (GBPs), including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses. Glycomics, like proteomics, has emerged as an important area of study in the postgenomics era. Glycans are essential for recognition, adherence, motility, and signaling in living systems, and GBPs play a key role in decoding glycan information by recognizing and binding to glycosylated proteins and lipids. However, the complexity of GBP-ligand interactions and the lack of well-defined glycan libraries and efficient screening methods have limited the analysis of GBP specificity and their biological roles. To address these challenges, the development of reliable and efficient tools for analyzing GBP specificity is needed. In recent years, various approaches for constructing glycan arrays for GBP specificity analysis have been developed. These approaches differ in the type of glycans and the method of display. Some use noncovalent attachment to plastic or nitrocellulose membranes, while others use covalent attachment to plastic, gold, or glass. The glycans displayed range from a limited number of exemplary structures to libraries of defined glycans, proteoglycan fragments, and microbial polysaccharides. This study describes a glycan array format that uses standard robotic printing technology to create a diverse glycan array with demonstrated applicability for profiling the specificity of a wide variety of GBPs. The array uses commercially available amine-reactive NHS-activated glass slides, which allow rapid covalent coupling of amine-functionalized glycans or glycoconjugates. The array was developed using a glycan library comprising over 200 synthetic and natural structurally defined terminal sequences of glycoprotein and glycolipid glycans, which could be readily modified to contain amino-functionalized linkers for covalent coupling to NHS-activated glass slides. The array was demonstrated to be useful for analyzing most major classes of GBPs, including mammalian lectins, plant lectins, antibodies, viral and bacterial lectins, and intact viruses. The array was used to analyze the specificity of various GBPs, including plant lectins, human GBPs, and bacterial and viral GBPs. The results showed that the array is highly effective for profiling GBP specificity and revealing unique aspects of fine specificity not previously recognized. The array has potential applications in therapeutic developments and screening for glycan-specific antibodies.