Background Measurement of iodine concentration from dual-energy or spectral computed tomography (CT) provides useful diagnostic information especially in patients suffering from malignant tumors of various origins. Purpose The purpose of this study was to systematically investigate the accuracy of the measurement of iodine concentration, focusing on potential influencing factors and assessing its suitability for routine clinical use. Material and Methods First, a 3D-printed cylindrical phantom was used to assess reliability of dual-energy CT-based iodine concentration measurement. Second, a semi-anthropomorphic phantom was used to evaluate the potential impact of positional variation of the target volume as typically seen in clinical scans. Finally, a reference vial was placed on the body surface of 38 patients undergoing abdominal dual-energy CT to analyze correlations between applied doses and patient diameters. Results The position of the target volume within the cylindrical phantom and the applied dose level significantly influenced the magnitude of measured iodine concentrations ( P < 0.001). We also found a significant difference in accuracy depending on target volume position in the semi-anthropomorphic phantom ( P = 0.028). In patient scans, we observed an error of 19.6 ± 5.6% in iodine concentration measurements of a reference and significant, moderate to strong, negative correlations between measured iodine concentration, maximum patient diameter, and applied dose (maximum sagittal diameter: r = −0.455, P = 0.004; maximum coronal diameter: r=−0.517, P = 0.001; CTDIvol: r = −0.385, P = 0.017) Conclusion Dual-energy CT-based iodine concentration measurement should be interpreted with caution. In clinical examinations, placement of a reference vial could be a potential solution to relativize errors.