The determination of glycosylation sites and oligosaccharide heterogeneity is key toward understanding the biological roles of glycoproteins. There are no previous methods that can reliably detetermine the site of glycosylation and the glycan heterogeneity at specific sites. We have developed a procedure for the determination of glycosylation sites and oligosaccharide heterogeneity in glycoproteins. The method is based on a combination of nonspecific proteolysis, deglycosylation, and high mass accuracy mass spectrometry analysis. The glycoprotein was proteolytically degraded using a nonspecific protease into glycopeptide fragments. The exact peptide mass was calculated by subtracting the glycan mass from the observed glycopeptide mass. The amino acid sequence of a matched peptide mass was identified from the protein database. A computer program, GlycoX, was developed in MATLAB to aid in the determination of the glycosylation sites and oligosaccharide heterogeneity in glycoproteins.