<p>Trace-level (ng/L) measurements of iodine’s isotopic (¹²⁹I and ¹²⁷I) and chemical species distributions are needed for an accurate understanding of subsurface radioiodine migration at former plutonium production complexes such as the Hanford and Savannah River sites. A previously developed method that uses ion chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) can measure speciated forms of ¹²⁹I at levels below the drinking water standard (~ 5.6&#xa0;ng/L), but an interference from molybdenum (Mo) prevents the assay from quantifying the total ¹²⁹I concentration in many relevant sample matrices. In this work, a preparative cation exchange method was developed to eliminate the Mo interference. The method requires little sample volume, is simple and fast to perform, and was shown to recover 96–99% of ¹²⁹I in test solutions containing approximately 20&#xa0;µg/L Mo and 2–4&#xa0;µg/L of either ¹²⁹I⁻ or ¹²⁹IO₃⁻. Method efficacy was further demonstrated by processing 11 groundwater and surface water samples with interfering levels of Mo from the Hanford and Savannah River sites. Total ¹²⁹I concentrations in the processed samples ranged from approximately 20–1175&#xa0;ng/L and generally agreed well with values independently obtained by radiochemistry methods. The sample preparation method enables quantification of total ¹²⁹I by ICP-MS in complex samples and provides a convenient approach for characterizing radioiodine distributions at contaminated nuclear waste sites.</p> Graphical abstract <p></p>