Purpose Erythropoietin (EPO) is a 165 amino acid protein that promotes the proliferation of erythrocytic progenitors. A decrease in endogenous EPO production causes anemia that can be treated with recombinant Human EPO (rHuEPO). Objective To ensure the safety and efficacy of the rHuEPO, manufacturers must use analytical methods to demonstrate similarity across batches and between different products. To do this they need reference standards to validate their equipment and methods. Method We used peptide mapping, size-exclusion chromatography, glycoprofiling, and isoelectric focusing to analyze a rHuEPO reference standard. Results Characterization demonstrates that our rHuEPO reference standard meets the criteria for quality. Conclusion The rHuEPO reference standard is fit for purpose as a tool for validating system suitability and methods.