ABSTRACT.- An enhanced protocol for the evaluation of in vivo biological activity of recombinant human erythropoietin (rhEPO) assay in mice is described, following European Pharmacopoeia (Ph. Eur.) version 10.0 guideline and optimized by applying the principles of Replacement, Reduction and Refinement (3Rs) in animal experimentation. In Laboratory Animal Science, one of the main principles is to optimize the experimental protocols in order to achieve the best results with the lowest number of animals and refine the procedures to avoid unnecessary suffering. The main objective of this protocol is to comply with international guidelines for rhEPO evaluation, applying refinement on procedures and reduction in animal use. Some of the features included in this protocol are the increase in number of rhEPO batches tested simultaneously against an international standard, leading to a substantial reduction in the number of animal's used, and refinement on animal handling techniques for subcutaneous drug administration and blood withdrawal. The implemented improvements were validated by reticulocyte estimation to ensure compliance with international criteria established for this trial and institutional quality management system. © 2023, Brazilian Society for Laboratory Animal Science. All rights reserved.