The primary objective of this study was to develop a simple method that can be used to extract the more readily mobilizable iodine species from soil while at the same time minimizing the transformation between IO3− and I−, the two most commonly found iodine species in the soil. High performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was used for iodine speciation analysis in soil. A sample pre-treatment process based on alkaline extraction in the presence of EDTA was optimized for the extraction of these iodine species. Good results were achieved using 10% ammonium hydroxide (v/v) and 0.5 mM EDTA where the transformation between IO3− and I− was clearly minimized. The method was applied to several soil reference materials. Recoveries of IO3− and I− spiked into these materials ranged from 85 to 115%. The most suitable sample collection and soil preparation techniques were also evaluated in this work.