A reverse phase high performance liquid chromatographic method for the simultaneous determination of cholesterol, α-tocopherol, retinol, retinal and retinoic acid in silicone oils, used as vitreous substitutes in eye surgery, is reported. The mixture components are removed from the matrix with recoveries close to 97% by solid-phase extraction with silica cartridges and subsequently separated on a C8column, using two different mobile phases consisting of acetonitrile/ammonium acetate and methanol/water that are passed sequentially.