The normocythemic mice bioassay was validated for the potency evaluation of the recombinant human erythropoietin (rhEPO) against the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The bioassays were performed in 8-week-old female BALB/c mice, which received multiple daily injections of standard or sample solutions (3 + 3), for 4 days. The blood sampling was performed 24 h after the last injection and the reticulocytes were counted by automated flow cytometry. Method validation investigated parameters such as linearity, precision, accuracy, specificity, and robustness, giving results within the acceptable range. The dose-response curve was linear in the concentration range of 1-64 international units (IU)/mL, and the value of the determination coefficient (r2) was 0.9708. The bioassay was applied for the potency evaluation of rhEPO pharmaceutical products containing alfa or beta forms, expressed in different cell lines, giving biological potencies within 82.79 and 119.70% of the stated potency. The precision index calculated by the weight for the independent assays was >247. The results demonstrated the validity of the bioassay for the potency assessment of pharmaceutical formulations contributing to ensure the therapeutic efficacy of the biological medicine.