This work presents an analytical methodology based on a "Quick, easy, cheap, effective, rugged, and safe" (QuEChERS) extraction and liquid chromatography-tandem mass spectrometry detection (LC-MS/MS) for the simultaneous determination of 35 psychoactive substances in fresh mussel samples. The compounds investigated include illicit drugs, new psychoactive substances, commonly prescribed psychiatric pharmaceuticals, and caffeine. The methodology was validated in terms of recovery (relative recoveries 77-118%), repeatability (relative standard deviation values <20%), and sensitivity (limits of detection and quantification <2 ng/g fresh weight (f.w.) and <6.7 ng/g f.w., respectively, for most compounds). The method was applied to the analysis of 15 samples, covering both commercially available mussels purchased from local food markets and wild fresh mussels collected in the Northeast coast of Spain in the Mediterranean Sea. Only one sample corresponding to wild mussels was found to contain 2 of the target analytes, namely, sertraline (1.5 ng/g f.w.) and caffeine, (12.8 ng/g f.w.).