Aim To establish a simplified method for extraction of lipids from high density lipoprotein (HDL), qualify and quantify lipids by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods The acidic methanol precipitation combined with ultrasonic extraction method was used to extract lipids, and the supernatant after centrifugation was analyzed directly by LC-MS/MS with multiple reaction monitoring mode. Results The acidic extraction method was effective and time saving, and the established LC-MS/MS method using lipids standards could simultaneously quantify more than 30 kinds of lipids, including sphingolipid, sphigomyelin, ceramide and oxidized phosphatidylcholine.Conclusions Acidic methanol precipitation combined with ultrasonic extraction can effectively isolate lipids from HDL, and the extract after centrifugation can be determined by LC-MS/MS directly. This method is effective with good reproducibility, and is suitable for quick quantitative analysis of a great deal of biological liquid samples.