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High-throughput method for stimulant NPS determination in biological samples.Ultra-rapid, simple, low solvent-use, economic.GC–MS analysis in SCAN mode.Possible reprocessing of datafiles to uncover further analytes.In the last few years an increasing number of new psychoactive substances (NPS), with different chemical structures (of which 37% are stimulants), have been released into the illicit drug market. Their detection and identification in biological samples is hence of great concern.The aim of this work was to develop a high-throughput and rapid method for the determination of different classes of stimulants (amphetamine-type stimulants, cathinones, phenethylamines and ketamine analogues) from blood and urine samples using GC–MS.The proposed method allows the almost simultaneous derivatization and extraction of analytes from biological samples in a very short time, by using hexyl chloroformate as derivatization agent. The extraction of analytes was performed by Dispersive Liquid Liquid Microextraction (DLLME), a very rapid, cheap and efficient extraction technique that employs microliter amounts of organic solvents.The chromatographic method allowed for the separation of 26 stimulants including positional isomers (3-MMC and 4-MMC). The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (LODs) ranging between 1 and 10 ng/mL, limits of quantification (LOQs) between 2 and 50 ng/mL, selectivity and linearity (5–1000 ng/mL).The method was then applied to real samples from forensic cases, demonstrating its suitability for the screening of a wide number of stimulants in biological specimens.