Oxidative stress has been proposed as one of the potential causes for infertility in men. Retinol and α-tocopherol have an important role in the spermatozoa defences against oxidative stress. A method is described here for the simultaneous determination of retinol and α-tocopherol in human seminal plasma with a suitable sample preparation procedure to prevent retinol and α-tocopherol degradation. After adequate sample preparation, the samples were determined by reversed-phase column chromatography with UV detection. The analytical performance of this method was satisfactory. The intra-assay and inter-assay coefficients of variation were below 10%. The recoveries were as follows: 90.7% (CV 8.1%) for retinol and 98.2% (CV 4.8%) for α-tocopherol. No significant differences in both retinol and α-tocopherol concentration between the smokers and nonsmokers (15 ± 7 nm and 1.86 ± 0.29 μm versus 15 ± 6 nm and 1.93 ± 0.45 μm) were found. A selective high-performance liquid chromatography method for the determination of retinol and α-tocopherol in human seminal plasma was developed.