Erythropoietin is a glycoprotein hormone that is responsible for regulating the synthesis of red blood cells. Recombinant human EPO (rhEPO) is the first cloned hematopoietic growth factor which is widely applied in therapeutic treatments especially for anaemia. Unfortunately, the ability of rhEPO to enhance haematopoiesis which increases the tissue oxygenation has led to its abuse in sports. The identification of rhEPO becomes very challenging due to its high similarity with endogenous EPO. Moreover, EPO and rhEPO have high diversity of glycoforms that have different but overlapped pI. Currently, the standard procedure to detect EPO abuse in urine and blood samples is still utilising gel-based tools which have consistency and sensitivity issues1 . Glycoproteomic mass spectrometry on the other hand is the new approach in the search for biomarkers2 to differentiate recombinant EPO from the endogenous one. Despite the high potential of glycoproteomic approach to be the alternative technique in anti-doping analysis of EPO, the current methodology is very time-consuming. In this study, several established proteolytic digestion methodologies were compared in order to shorten and optimise the glycoproteomic procedure. Our LTQ-Orbitrap liquid chromatography-mass spectrometric analysis coupled with PEAKS de novo sequencing and database matching on digested recombinant and endogenous EPO was able to achieve optimum protein sequence coverage. The mass spectrometric data of glycopeptides obtained were then used to identify the glycoproteomic biomarker of EPO. Preliminary differentiation of a number of recombinant EPO biosimilars will be presented.