The disturbed cholesterol metabolism was associated with various disease states.Thus, recently, study on cholesterol metabolism have become have become important tasks in the clinical fields.In this study, efficient methods for cholesterol analysis were developed by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry as its tert-butyldimethylsilyl derivative.The two methods were linear (r ≥ 0.995) with good precision and accuracy, allowing quantitative analysis of cholesterol in biological fluids, which were complementary of each other.The present methods will be useful for screening and monitoring of cholesterol in biological fluids.