Chemicals that end up in the water cycle can negatively impact water quality and thereby the aquatic environment. In the Netherlands, drinking water is primarily prepared from surface water and groundwater. To protect the environment and drinking water sources, it is important to adequately monitor the water quality and identify contaminants of emerging concern (CECs). Current European legislation on water quality assessment dictates the use of targeted chemical analysis to analyze a list of priority substances in water bodies. By only measuring a limited list of compounds, however, environmental risks and adverse effects on water quality may be underestimated. To improve this assessment, effect-based bioassays are applied more and more. Bioassays measure the combined potency of all (known and unknown) bioactive compounds in a sample that affect the same endpoint. Effect-directed analysis (EDA) combines chemical analyses and bioassays into one platform to identify CECs, to prioritize them for future monitoring. Previously, such a platform was developed in the High-Throughput EDA project (2013 – 2017, funded by NWO-STW). In this project, the Division of Bioanalytical Chemistry and the VU Department of Environment & Health of Vrije Universiteit Amsterdam collaborated with Het Waterlaboratorium. The developed platform was implemented at Het Waterlaboratorium for monitoring applications outside an academic context. In the HT-EDA project, bioassays that monitor mutagenic and endocrine disrupting activity were applied for the identification of compounds with EDA. Furthermore, the platform allowed for high-resolution fractionation, simplifying the composition of mixtures in fractions in well plate format. The identification success rate was limited, however, which could be (partly) attributed to a lack of fast identification strategies. Also, there was a need to identify CECs with other toxicological endpoints relevant to the water cycle, such as the toxicity of antibiotics. The work in this thesis was carried out as part of the RoutinEDA project, which was embedded in the CEC-partnership, a research program funded by the Dutch Research Council (NWO), STOWA, TKI Water Technology, and KWR Water Research Institute. Chapter 1 describes the background and the aims of this thesis. Here, information on chemicals that threaten water quality and water quality assessment strategies are provided. Further, it is described how targeted chemical analysis and suspect and nontarget screening approaches are applied in the identification of CECs. The principle of EDA is presented in detail, together with novel toxicological endpoints relevant to the water cycle that were addressed in this work. The aims of this thesis were to: (1) Expand the existing EDA platform with an antibiotics bioassay, targeting antimicrobial compounds as a toxicological endpoint (2) Enhance the identification success rate of CECs (3) Identify CECs with glucocorticoid and/or antimicrobial activity in water samples (4) Improve the EDA platform in efficiency and use allowing for routine application by monitoring laboratories Chapter 2 describes the development of a high-throughput bioassay for the screening of antibiotics in environmental samples. In Chapter 3, a data processing workflow for EDA studies was developed to improve the identification process of CECs using suspect and nontarget screening. Chapter 4 describes the identification of antimicrobial CECs in water sample extracts that were sampled around two WWTPs by using EDA. In Chapter 5, six Dutch WWTP effluents were subjected to EDA for the identification of CECs with glucocorticoid and/or antimicrobial activity. Finally, Chapter 6 discusses the overall findings of this thesis and provides future perspectives to improve the EDA platform.