The qualitative and quantitative determination of cholesterol and 7-dehydrocholesterol (7-DHC) in plasma as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome by liquid chromatography/particle beam interface-mass spectrometry (LC/PB-MS) is presented. Baseline separation of cholesterol and 7-DHC is achieved on a silica column with hexane+ethanol (99: 1 v/v) as mobile phase within a 10 min analysis. Recoveries of cholesterol and 7-DHC in a simple two-phase extraction system are nearly 100%. The absolute limit of detection using LC/PB-MS is approximately 10 ng. The method presented allows extraction, analysis and quantification of cholesterol and 7-DHC within 15 min without the necessity of sample derivatization.