Abstract: A method for screening 40 drugs in human hair by high performance liquid chromotography tandem mass spectroscopy (HPLC-MS/MS) has been established and applied for the detection of real hair samples. In this method, the linearity relationship was studied using the standard samples of 40 drugs, and human hair samples, which were ground into powder using a freeze grinder, extracted in methanol at 70℃ under ultrasonic extraction for 2 h. The concentrated extract was reconstituted to 100 μL by methanol for HPLC-MS/MS analysis. Result shows the recovery rate for 40 drugs of the method was between 77.3%~93.3%, the correlation coefficient was above 0.942 in the linear range of 9~770 ng/g, and the limit of detection was 0.01~0.8 ng/g. The method is rapid, sensitive, accurate, and can be used to the qualitative and quantitative analysis of 40 drugs in human hair.