A highly sensitive and specific indirect competitiveenzyme-linked immunosorbent assay (ELISA) for thedetermination of diclofenac in water samples was developed.With pure water, the limit of detection (LOD, S/N ) 3)and IC50 were found to be 6 ng/L and 60 ng/L, respectively.The analytical working range was about 20-400 ng/L.Highest cross-reactivity (CR) of 26 tested pharmaceuticals,metabolites, and pesticides was found for 5-hydroxydiclofenac(100%). Other estimated values were well below4% and, therefore, are negligible. The assay was appliedfor the determination of diclofenac in tap and surface watersamples as well as wastewater collected at 20 sewagetreatment plants (STPs) in Austria and Germany. Humicsubstances were identified as main interference in surfacewater. Wastewater samples which were only submittedto filtration and dilution yielded about 25% higher diclofenacconcentrations using the ELISA compared to GC-MS.However, the ELISA turned out to be a simple, inexpensive,and accurate method for the determination of diclofenacboth in influent and effluent wastewater after rather simplesample preparation, i.e., filtration, acidification, andreadjustment to neutral pH-value, and at least 10-folddilution with pure water.