Carbohydrate antigens are important targets for the immune system, but identification of key glycan antigens is challenging. Direct analysis of glycomes by mass spectrometry is difficult, and detection reagents, such as monoclonal antibodies and lectins, are only available for a small subset of glycans. An alternative approach involves profiling serum anti-glycan antibody populations to identify unique antibodies or changes in antibody subpopulations. Glycan microarray technology allows rapid evaluation of hundreds to thousands of antigen-antibody interactions in a single experiment. This high-throughput format is particularly useful in profiling complex anti-glycan antibodies in serum. Here we elaborate the use of this technology to explore clinically relevant carbohydrate antigens by profiling serum anti-glycan antibodies. Detailed protocols from glycan microarray fabrication to microarray binding assays and analysis of microarray data are presented.