Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots.