The analysis of the N-glycan portions of glycoproteins has become important for the detection of various diseases involving altered glycosylation patterns, including cancer and congenital disorders of glycosylation, and in the quality control of therapeutic recombinant glycoproteins. Here in this chapter we give detailed analysis procedures necessary for the complete structural elucidation of any N-glycan mixture found in naturally occurring and recombinant glycoproteins. The protocols include monosaccharide composition analysis on the intact glycoprotein, the enzymatic release of N-glycans from glycoprotein and their separation from O-glycopeptides and peptides, and the structural characterization of the glycans by exoglycosidase digestions, methylation ("linkage") analysis, and mass spectrometry/tandem mass spectrometry of permethylated glycans using matrix-assisted laser desorption and electrospray ionization methods.