A method based on CZE for the determination of glutamic acid, glycine, and alanine in a biopharmaceutical formulation containing recombinant human erythropoietin was developed. The separation was achieved within less than 5 min, using a fused-silica capillary column (55 cm x 50 m id) and 30 mmol/L phosphate buffer at pH 11.5, containing 0.6 mmol/L CTAB and 10% v/v methanol, as BGE solution. Applied potential of -25 kV, temperature of 15 degrees C and hydrodynamic injection time of 15 s, at 50 mbar, were employed. The detection of the analytes was carried out without any derivatization reaction, at 220nm using an UV-DAD detector. Linear ranges from 50 to 2500 mg/L and quantification limits of 40, 39, and 37 mg/L were obtained for glutamic acid, glycine, and alanine, respectively. Sample preparation required only a dilution step. Considering peak area and migration time values, the method presented good repeatability (RSD <1.7%; n = 9) and intermediate precision (RSD <1.0%; n = 6). Recovery evaluation using a commercial sample led to values between 97.5 +/- 5.2% and 101.5 +/- 4.6%, demonstrating the feasibility of the method, which was successfully applied in the quantification of the amino acids of interest in biopharmaceutical samples.