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Herein we present the data for a newly developed immunoassay for the determination of amoxicillin (AMX) in water samples using a commercially available monoclonal anti-AMX antibody. During optimization and testing of the assay, we observed a significant decrease of the C-value (IC50) of the ELISA calibration curves with aged standard solutions. Running the assay with standard solutions of synthesized AMX hydrolysis products revealed that this decreased C-value could be attributed mainly to the hydrolysis product amoxicilloic acid. Therefore, the limit of detection of the ELISA for AMX determination in water could be enhanced by either hydrolyzing the samples or testing for amoxicilloic acid as marker substance.