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Hydroxytriazines are environmental decomposition products of s-triazine herbicides. Enzyme-linked immunosorbent assays (ELISAs) in various formats for the detection of hydroxypropazine, hydroxyatrazine, and hydroxysimazine were developed. 6-{4-Methoxy-6-[(1-methylethyl)amino]-1,3,5-triazin-2-yl}aminohexanoic acid was used as the immunizing hapten. One of the resulting ELISAs has IC50 values of 0.002, 0.004, and 0.017 μM (0.4, 0.8, and 3.1 ppb) for hydroxypropazine, hydroxyatrazine, and hydroxysimazine, respectively. This ELISA utilizes a coated antigen format with a simazine−mercaptopropanoic acid hapten conjugated to conalbumin as the coating antigen. The assay parameters are unchanged at pH values between 5.0 and 8.0 and at ionic strengths from 0.13 to 1.0 M phosphate-buffered saline. Methanol does not affect the assay up to 13%, whereas acetonitrile and acetone cause a decrease of sensitivity at 3.1%. Groundwater fortified with hydroxyatrazine was analyzed according to this method. Good correlations with spike levels were observed, making this assay potentially valuable for the screening of groundwater samples containing parts per trillion levels of hydroxytriazine contamination. Keywords: s-Triazine; hydroxytriazine; hydroxyatrazine; hydroxysimazine; hydroxypropazine; ELISA; enzyme immunoassay; herbicide metabolites