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Sample preparation was done by protein precipitation with acetonitrile in 96\u2010well format and aspirin was used as the internal standard. The 100\u00a0\u00b5L sodium thiosulfate solution (5\u00a0g\u00a0L<jats:sup>\u22121<\/jats:sup>) was added to 100\u00a0\u00b5L plasma sample before protein precipitation, to convert the total free iodine in plasma to iodide (I<jats:sup>\u2212<\/jats:sup>). Separation was performed on a C<jats:sub>18<\/jats:sub> column (200\u00a0\u00d7\u00a04.6\u00a0mm i.d., 5\u00a0\u00b5m). The mobile phase consisting of a mixture of water phase (containing 10\u00a0mmol\u00a0L<jats:sup>\u22121<\/jats:sup> 18\u2010crown\u20106 ether, 5\u00a0mmol\u00a0L<jats:sup>\u22121<\/jats:sup> octylamine and 5\u00a0mmol\u00a0L<jats:sup>\u22121<\/jats:sup> sodium dihydrogen phosphate, pH adjusted to 6.0 with phosphoric acid) and acetonitrile in the ratio 70:30 (v\/v) was delivered isocraticly at a flow rate of 1.0\u00a0mL\u00a0min<jats:sup>\u22121<\/jats:sup>. The method was sensitive with a lower limit of quantification of 0.005\u00a0\u00b5g\u00a0mL<jats:sup>\u22121<\/jats:sup>, with good linearity (<jats:italic>r<\/jats:italic><jats:sup>2<\/jats:sup>\u00a0&gt;\u00a00.9990) over the linear range of 0.005\u20132\u00a0\u00b5g\u00a0mL<jats:sup>\u22121<\/jats:sup>. All the validation data, such as linearity, accuracy and precision, were within the required limits. The method was successfully applied to study the pharmacokinetic of PVP\u2013I in rabbits after vaginal administration. 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