High-performance chromatography method was developed and validated for essaying α-tocopherol and cholesterol in cryopreservation media. Chromatographic separation was performed on isocratic system, using a C-18 column. The mobile phase was composed of a mixture of methanol: acetonitrile: water 68:28:4 (v/v/v), using a flow rate of 1.5 ml/min and 20 μl injection volume, at a wavelength of 208 nm. The method was validated according to ICH guidelines. The method proved to be specific, accurate, precise and linear with correlation coefficients greater than 0.996 over a wide concentration range of both analytes. Vitamin E and cholesterol presented limits of detection of 0.002 mg/ml, 0.026 mg/ml and limits of quantitation of 0.006 mg/ml, 0.086 mg/ml, respectively. This method shows simple, rapid, high precision and accuracy and offers the advantage of simultaneous assay of vitamin E and cholesterol (alone, in cyclodextrins complexes or in liposome loaded) on semen cryopreservation media.