Glycosylation is one of the most common post-translational modifications in proteins. Current methods for glycan analysis are generally based on multiple preparation processes to separate glycans. However, glycans are continuously lost and the difficulty for accurate quantitative analysis is increased in the procedure. Here, a filter aided sample preparation-based total N-linked glycans from the glycoproteins enrichment and separation method (N-glycan-FASP-T) was developed using ultrafiltration units according to the molecular mass difference among the glycans, the impurities and proteins. The enriched glycans were characterized and confirmed by the MALDI-TOF/TOF-MS. A total of 23 distinctive N-linked glycans were characterized from human serum.