Glycosylation is highly sensitive to the biochemical environment and plays a key role in development and disease manifestation. Moreover, glycan biosynthesis depends on several highly competitive processes; thus, variations in the concentration of specific glycosyltransferases produce different products. For this reason, monitoring changes in glycosylation may be a more specific and sensitive approach to biomarker discovery and possibly disease diagnosis. Glycans in serum are of particular interest as approximately half of all proteins are glycosylated. We have developed the methods for profiling the glycans in human serum to identify glycan biomarker. Global release methods were used including chemical and enzymatic to access O-linked and N-linked glycans, respectively. Glycans were released from the culture medium of various cancer cell lines, in control sera, and in cancer patients and isolated using solid phase extraction (SPE) with a porous graphitized carbon. The SPE fractions were analyzed by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS). Glycan compositions were determined based on accurate masses and tandem mass spectrometry. Glycosylation changes between control and patient group were monitored. Several glycans were identified as potential markers for ovarian, breast, and prostate cancer. In short, direct glycan analysis of human serum without any protein identification represents a new and innovative approach to disease marker discovery.