Résumé : Epoetin beta is a recombinant human glycoprotein used to correct anemia in dialysis patients. When administered with dosing 30 000 to 60 000 IU per week, epoetin beta has recently shown an interest in prevention and treatment of anemia induced by cancer chemotherapy in adults with solid and liquid tumors. The development of a sensitive and specific analytical method is necessary to individualize and optimize treatments in non-responding patients, and in responders to determine effective maintenance doses. We have developed a new strategy based on mass spectrometric analysis of epoetin beta isolated after depletion of major human plasma proteins, and digested by trypsin before chemical labelling used for quantification. The first step of enrichment of epoetin beta from human plasma uses affinity chromatography to remove the seven major plasma proteins (HU-7, Agilent Technologies). Epoetin beta enriched fraction is desalted and purified by reversed phase chromatography before tryptic proteolysis. To quantify the erythropoietin, tryptic peptides obtained are labelled on lysine residues (68.03 Da increment). Epoetin beta tryptic peptides labelled by the same reagent bearing an isotopic label (72.06 Da increment) are used as internal standards for absolute quantification. In a first development phase, analysis was made in MALDI ionization mode. Analysis with an ESI-Q-TOF spectrometer identified three labelled peptides, one of which was used for quantification. This quantitative analysis strategy has two major interests, because it does not require a synthetic labelled reference peptide and this methodology can be used for the analysis of other plasma protein drugs.