A novel, simple and fast method based on a quadrupole ion trap (QIT) and a time-of-flight mass spectrograph was developed for the glycosylation site and glycan structure analysis of glycoprotein by in-solution digestion and in-gel digestion which did not need to enrich and label the glycopeptide. Horseradish peroxidase (HRP) and RNase B were utilised to optimize the conditions of sample pre-processing. The effects of the conditions were investigated separately and the optimal conditions were: heat denaturation of RNase B, chemical denaturation of HRP, RNase B digestion with endoproteinase Lys-C, HRP digestion with trypsin, 12 - 16 h of digestion, 50% acetonitrile-5% trifluoroacetic acid of the extraction solution, sandwich spotting method of the sample.