The present work describes the investigation of the formation of oxidation by-products of the iodinated X-ray contrast medium (ICM) iomeprol during ozonation in water treatment. Bench-scale investigations revealed that ICM can be partly oxidized during ozonation processes, whereas the ionic diatrizoic acid showed the lowest reactivity. Iomeprol, as a representative of ICM, was not fully mineralized during ozonation. Thus, unknown oxidation by-products were formed. Aqueous solutions of iomeprol were treated by ozonation in order to assess the formation of oxidation by-products. The by-products were characterized by different liquid chromatography methods including detection of single-stage mass spectra, product ion mass spectra, and induced in-source fragmentation for analysis of iodine containing oxidation by-products. Aldehyde and carbonyl containing compounds were proposed to be among the stable by-products. A derivatization step confirms that the aldehyde and carbonyl moieties are major functional groups in oxidation by-products of iomeprol. Furthermore, oxidation by-products of iomeprol were detected at the outlet of an ozone reactor at a full-scale waterworks. However, the toxicological relevance of the by-products is a major future research tasks.