For the first time a comprehensive two-dimensional liquid chromatography (LC × LC) system coupled with a high resolution time-of-flight mass spectrometer (HR-ToF MS) was developed and applied for analysis of emerging toxicants in wastewater effluent. The system was optimized and validated using environmental standard compound mixtures of e.g. carbamate pesticides and polycyclic aromatic hydrocarbons (PAHs), to characterize the chromatographic system, to test the stability of the retention times and orthogonality. Various stationary phases in the second dimension were compared for the LC × LC analysis of silicon rubber passive sampler extracts of a wastewater effluent. A combination of C18 and Pentafluorophenyl (PFP) was found to be most effective. Finally, the hyphenation of LC × LC with HR-ToF MS was optimized, including splitter settings, transfer of data files between the different software packages and background subtraction using instrument software tools, after which tentative identification of 20 environmental contaminants was achieved, including pesticides, pharmaceuticals and food additives. As examples, three pesticides (isoproturon, terbutryn and diazinon) were confirmed by two-dimensional retention alignment.