The glycosyl modification on protein therapeutics is considered to be a very important attribute due to its strong effect on quality, safety, and potency. Thus, systematic approach is absolutely required for measurement and control of glycosylation from the drug development to post-approval batch release. We have developed stepwise strategies for glycome quality assessment during drug design and manufacture in the level of intact protein, glycopeptide, and glycan. Intact protein analysis was employed for detailed characterization of highly complex micro—heterogeneity, including various glycofrom profiles by high-resolution native mass spectrometry. Site-specific glycosylation with micro & macro glycan heterogeneity can be achieved by glycopeptide analysis which combines molecular size fractionation, multiple proteolysis, and PGC nanoLC MS/MS. This approach could be widely applied for high-quality analytical characterization of glycosylation, starting from early drug development to final lot releas.