As one of the most common post-translational modifications, protein N-glycosylation is involved in most important biological processes. Structural changes of the protein linked N-glycans have close correlations with the occurrence and progression of various diseases. Therefore, efficient detection of these disease-associated aberrations is of great significance for the development of new diagnostic biomarkers and therapeutic drug targets. In this work, we developed a method for convenient and effective N-glycan detection by coupling condition controlled chemical derivatization with MALDI-TOF MS. Due to the high reaction efficiency and enhanced hydrophobicity, improved identification scales were obtained for N-glycans from both asialofetuin and human plasma compared with the direct analysis.