Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were compared for analyzing microcystins in water. ELISA results of microcystin‐LR spiked into raw water samples were close to the spike concentrations, but method variability was ±25%. However, ELISA‐derived microcystin‐LA concentrations were two to three times higher than the spike concentrations obtained using the kit‐provided microcystin‐LR standards, indicating the need for variant‐appropriate ELISA standards. LC/MS/MS results agreed with spike concentrations for all variants in reagent water, but matrix suppression was observed in some raw waters. In bench‐scale studies, ozonated microcystins generated low‐level positive responses by ELISA and a protein phosphatase inhibition assay, even though microcystins were not detected by LC/MS/MS. These findings indicate that ELISA results—particularly in treated water—should be interpreted with caution because of the possibility of false‐positives, relatively high variability, and differential detection of some variants.