Protein glycosylation plays an important role in multiple cell functions, and aberrations of protein glycosylation are associated with various malignancies including cancer. In this chapter, we provide a detailed protocol for MALDI MS analysis of permethylated N-glycans extracted from human serum proteins. The protocol includes procedures for N-glycan purification and in-solution permethylation, structural elucidation of permethylated N-glycans by MALDI-QIT-TOF MS, and construction of indices to quantify levels of certain types of glycosylation, such as fucosylation, which may serve as a potential disease biomarker.