Erythropoietin (EPO) is a glycoprotein with hormone activity that controls the production of red blood cells in the bone marrow. Recombinant human EPO is produced on a large scale in cell culture and therefore, available as therapeutic agent for treating anemia related to different diseases. It is also known as blood doping agent in endurance sports. Human EPO is an approximately ~30 kDa glycoprotein with one O- and three N-glycosylation sites. In pharmaceutical drug production, glycosylation heterogeneity of EPO is an important quality characteristic providing functionality as well as bioavailability of the therapeutic protein. Previous results have shown a direct positive correlation between the sialic content, increased half-life in the blood, and its bioactivity 1- 3. As such, it has been speculated that the potency of EPO may be increased by means of engineered glycosylation, such as the formation of highly branched sialylated N-glycans, introduction of additional glycosylation sites, or via metabolic engineering to provide more CMP-sialic acid for sialylation 1-3. Here, we describe a mass spectrometric approach including dedicated software tools to automatically identify O-linked glycosylation patterns of tryptic glycopeptides from human EPO of 2 different origins.