Phytotoxins, secondary metabolites synthesized by plants, are now recognized as a new category of environmental micropollutants. Only a limited number of phytotoxins have been detected and reported in terrestrial and aquatic environments, partially due to analytical challenges. In this article, a robust, reliable, and efficient approach, namely Source Supported Suspect Screening (4S), is introduced for high-throughput analysis of phytotoxins from their plant origin to the downstream environmental compartments. The approach was established on an ultrahigh-performance liquid chromatography electrospray ionization–quadrupole-time-of-flight-mass spectrometry (UHPLC–ESI–QTOF-MS) analytical platform where optimal sample preparation and chromatographic conditions were investigated and a new suspect screening protocol was suggested.