A rapid LC-MS/MS method for the targeted screening of 132 NPS in hair is described. Drugs include cathinones and synthetic cannabinoids, as well as amphetamine-type stimulants, piperazines and other hallucinogenic compounds. This method utilizes hair pulverization in acidified methanol followed by analysis using liquid chromatography coupled with tandem MS. The limit of detection varied from 0.001 to 0.1 ng/mg hair among the various analytes. The method was validated in terms of sensitivity, selectivity, repeatability and stability. The limit of reporting was set at 0.1 ng/mg of hair. The method was successfully applied to 23 medico-legal cases where NPS were detected in blood or where NPS use was suspected. The identified NPS included acetyl fentanyl, 25C-NBOMe, MDPV, PB-22 and AB-FUBINACA, allowing hair to be used where historical or retrospective information on use of NPS is sought. This technique has proven to be efficient for the one step extraction from hair of different classes of NPS in routine toxicological investigations; from unstable and volatile compounds, such as most of the cathinones, to hydrophobic compounds such as synthetic cannabinoids.