The identification of unknown toxicants in complex water samples such as wastewater cannot be achieved with target chemical analysis alone. Tools and technologies that reduce the complexity of the sample and identify the chemicals responsible for toxic effects are necessary. The present study focused on developing a diagnostic bioanalytical strategy that integrates toxicological and chemical analyses based on effect-directed analysis (EDA) in order to identify toxicants in complex wastewater samples. The workflow is based on combining targeted high-resolution mass spectrometry (HRMS) analysis, fractionation, and bioassays to elucidate the structure of toxicants. Several fractionation strategies were investigated including thin-layer chromatrography, and preparative HPLC. Also, a chip-based nano-ESI source coupled to Orbitrap analyzer  was used in the LC/MS fraction collector mode. This study highlights the importance of effect-directed analysis as an approach for the identification of unknown toxicants in complex water samples.