Glycosylation is an essential post-translational modification of protein, which is involved in many biological processes including protein folding, immune response, protein-protein interaction and pathogens. Glycan exhibits complexity and dynamic changes in not only compositions of monosaccharides but also the linkages of them. Mass spectrometry is a powerful approach that provides a systematic and high-throughput analysis of protein glycosylation. In this dissertation, the mass spectrometry techniques have been utilized to analyze therapeutic glycoproteins and discover novel biomarkers of colon cancer. Numerous analytical chemistry techniques have been applied, including hydrophilic interaction enrichment of glycopeptide, solid phase based identification of O-glycosylation site, 18O labelling of N-glycosylation site, stepped collision energy induced glycopeptide dissociation, HCD/ETD alternative dissociation and CID based multi stage mass spectrometry. These advanced techniques have been applied in this dissertation to comprehensively understand the glycosylation of Coagulation factor V, discover the potential glycoprotein biomarkers of colon cancer and elucidate the unrevealed structure of pneumococcal polysaccharide vaccine.