The ongoing appearance of new psychoactive substances on the black market of illegal drugs, as well as the lack of information on their influence on the human body, faces several challenges in their determination by standard analytical techniques. Moreover, the rapid metabolism of new psychoactive substances reveals in the absence of possibility in the identification of their native structures in biological fluids. This study presents a new screening method for determination 137 psychoactive substances including their metabolites. 'Dilute-and-shoot' method was chosen as the preferable sample preparation technique, and consisted of 1:5 dilution of urine specimens with the solution of acetonitrile and water (30:70) followed by electrospray ionization – liquid chromatography-tandem mass spectrometry analysis. The developed qualitative method was validated according to United Nations Office on Drugs and Crime requirements that included assessment of selectivity, limits of detection, precision, and stability. In addition, the presented method was tested on 50 certified positive urine specimens containing different drugs of abuse. The confirmatory analysis was performed using a high-resolution mass-spectrometry approach. The presented screening method provides the possibility of simultaneous determination of synthetic cannabinoids (96), opioid analgesics (16), stimulators (13), hallucinogens (5), benzodiazepines (5) and non-classified drugs (10) during one run. The validation assessments of the novel method have shown high rates of its specificity, selectivity, intra- and inter-day precision and stability with the limit of detection ranged from 1 to 5 ng?mL-1. At the same time, tests of 50 positive samples showed excellent applicability of the developed screening method for routine preliminary screening analysis in toxicological laboratories.