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New psychoactive substances are produced and marketed to mimic the effects of their illicit counterparts and to attempt to evade drug tests and prosecution. Here, we present the optimisation, validation and application of an analytical method using liquid chromatography–time‐of‐flight mass spectrometry to detect and quantify 37 new psychoactive substances and illicit substances in wastewater from South Wales, UK, using a targeted analysis method. Sample preparation was performed using solid‐phase extraction with Oasis HLB cartridges. The LC separation was performed using a YMC‐Triart Phenyl 450 bar column (12 nm, 5 μm, 100 × 3 mm) which provided good separation and resolution for all targeted analytes with a run time of 9 min. The method was validated using the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recovery and matrix effects. The method was then applied to influent wastewater samples collected from two wastewater treatment plants in Wales, UK. [ABSTRACT FROM AUTHOR]