A method by reverse phase High Performance Liquid Chromatography with spectrophotometric detection on UV range (HPLC-UV) simple and fast was developed and validated for determination of recombinant human Erythropoietin (rhEPO) in presence of human serum albumin (HSA) as stabilizer in three different presentations; 2000, 4000 and 10,000 international units (IU). A ballistic gradient was obtained for separation of both proteins of biopharmaceuticals at time lower than 5 min. The solvent system comprised: solvent A – deionized water (0.1% trifluoracetic acid v/v), solvent B – acetonitrile (0.05% trifluoracetic acid v/v). The stationary phase was an octyl silane, C8, analytical column from J. T. Baker®. The flow rate was 1.5 mL min− 1 , while the column and autosampler temperatures was kept at 60 and 5 ◦C, respectively. The injection volume was 100 μL and the wavelength monitored by detector was 214 nm. The method has been validated according to the following analytical figures of merit: selectivity, trueness, linearity, precision (repeatability and intermediate precision) and limits of detection and quantification. Moreover, additional studies as stability of sample solution, peak purity test and uncertainty of measurement were assessed. The present study used a reference standard of Erythropoietin (BRP) supplied by European Pharmacopoeia in addition to in-house reference material of Erythropoietin produced in collaboration between Bio-Manguinhos (FIOCRUZ/BRASIL), The National Institute of Quality Control in Health, INCQS (FIOCRUZ/BRASIL) and The Center of Molecular Immunology, CIM (CUBA).