Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-α, EPO-β and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25–50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop®) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-α, EPO-β and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-α/β and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 ± 0.3 kDa for EPO-α/β and 36.8 ± 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-α and EPO-β, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N- or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-α, EPO-β and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein. Copyright © 2005 John Wiley & Sons, Ltd.