This work reports a simple isocratic hydrophilic interaction liquid chromatographic (HILIC) method for the simultaneous quantification of iodixanol and of its related impurities C, D and E in drug substance. The chromatographic separation was carried out with a Kinetex™ HILIC column, using acetonitrile and formic acid aqueous solution (1.0 mmol/L, pH 3.2) (92:08, v/v) as eluent at a flow rate of 0.8 mL/min. The autosampler and column temperature were maintained at 20 °C and UV detection was set at 243 nm. The method was validated in accordance to the ICH guideline and employed for the analysis of two different lots of iodixanol drug substance. The developed method is presented as a valuable alternative to the current methods described in the USP monograph.