Approximately more than half of mammalian proteins are glycosylated. O-linked glycan, attached to protein via serine or threonine residue, is one of common post-translation modifications on proteins. Its main functions include maintaining the conformation of the protein connected, protecting it from proteolysis, and covering some antigenic determinant. Analysis of O-linked glycan structure of glycoproteins can contribute to a clearer understanding of glycoproteins and their functions. This study describes a new strategy, involving enrichment and separation of total O-glycan from the glycoproteins based on a filter assisted sample preparation method (O-glycan-FASP), which was developed using ultrafiltration units according to the molecular mass differences among the glycans and proteins. The glycans were characterized and confirmed by MALDI-TOF/TOF-MS. A total of 105, 29, 33 and 85 distinctive O-glycan were characterized from bovine submaxillary mucin (BSM), human cell, serum and urine respectively.