Recombinant human erythropoietin (rhEPO) has been extensively used as pharmaceutical product for treatment of anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics and in vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for its quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple enzyme digestion, hydrophilic interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high resolution and high accuracy (HR/AM) mass spectrometry on an Orbitrap. Totally, 74 intact glycopeptides from four glycosylation sites at N24, N38, N83 and O126 were identified with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR/AM data allowed relative quantification of glycoforms. Our results could be extended to the quality control of rhEPO or help to establish detection approaches for glycosylation of other proteins.