Because of its nutritional, anti-oxidative, and cryoprotective properties, human serum albumin (HSA) is an important ingredient of the culture and cryopreservation media for assisted reproductive techniques (ART) such as in vitrofertilization (IVF) and intracytoplasmic sperm injection (ICSI) procedures. Several tools are available for the determination of this serum protein in biological samples and pharmaceutical preparations, including colorimetric, electrophoretic, and immunological assays. However, because of inter-assay variability and accuracy problems of the above-mentioned assays, we have chosen to develop and validate a reverse phase (RP) high-performance liquid chromatography (HPLC) method to assess HSA content in ART-related media. Briefly, a gradient elution (a combination of acetonitrile/water, supplemented with 0.1% (v/v) trifluoroacetic acid) was used to separate samples on a C4 (n-butyl-coated silica) column. Two main peaks were observed at 4.970 and 8.715 min, representing the stabilizer N-acetyl-tryptophan (N-Ac-Trp) and HSA respectively. Validation of the method demonstrated that HSA can be determined in an accurate and precise manner, in a range between 0.4 and 25 mg ml⁻¹, without interference of matrix ingredients. The limit of detection (LOD) and lower limit of quantification (LLOQ) values were 0.128 and 0.386 mg ml⁻¹, respectively. In summary, this RP-HPLC method serves as a quality control for ART product release and stability studies. If required, the method can be easily adapted for assessment of HSA in biological samples and pharmaceutical preparations.