There are huge numbers of clinical specimens being stored that contain potential diagnostic marker molecules buried by the coexistence of high-abundance proteins. To utilize such valuable stocks efficiently, we must develop appropriate techniques to verify the molecules. Glycoproteins with disease-related glycosylation changes are a group of useful molecules that have long been recognized, but their application is not fully implemented. The technology for comparative analysis of such glycoproteins in biological specimens has tended to be left behind, which often leads to loss of useful information without it being recognized. In this chapter, we feature antibody-assisted lectin profiling employing antibody-overlay lectin microarray, the most suitable technology for comparative glycoanalysis of a trace amount of glycoproteins contained in biological specimens. We believe that sharing this detailed protocol will accelerate the glycoproteomics-based discovery of glyco-biomarkers that has attracted recent attention; simultaneously, it will increase the value of clinical specimens as a gold mine of information that has yet to be exploited.