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Objectives: Opposed to other spectral CT techniques, fat quantification in dual-layer detector CT (dlCT) has only recently been developed. The impact of concomitant iron overload and dlCT-specific protocol settings such as the dose right index (DRI), a measure of image noise and tube current, on dlCT fat quantification was unclear. Further, spectral information became newly available <120 kV. Therefore, this study's objective was to evaluate the impact of iron, changing tube voltage, and DRI on dlCT fat quantification., Material and Methods: Phantoms with 0 and 8mg/cm3 iron; 0 and 5mg/cm3 iodine; 0, 10, 20, 35, 50, and 100% fat and liver equivalent, respectively, were scanned with a dlCT (CT7500, Philips, the Netherlands) at 100kV/20DRI, 120kV/20DRI, 140kV/20DRI, and at 120kV/16DRI, 120kV/24DRI. Material decomposition was done for fat, liver, and iodine (A1); for fat, liver, and iron (A2); and for fat, liver, and combined reference values of iodine and iron (A3). All scans were analyzed with reference values from 120kV/20DRI. For statistics, the intraclass correlation coefficient (ICC) and Bland-Altman analyses were used., Results: In phantoms with iron and iodine, results were best for A3 with a mean deviation to phantom fat of 1.3±2.6% (ICC 0.999 [95%-confidence interval 0.996-1]). The standard approach A1 yielded a deviation of -2.5±3.0% (0.998[0.994-0.999]), A2 of 6.1±4.8% (0.991[0.974-0.997]). With A3 and changing tube voltage, the maximal difference between quantified fat and the phantom ground truth occurred at 100kV with 4.6±2.1%. Differences between scans were largest between 100kV and 140kV (2.0%[-7.1-11.2]). The maximal difference of changing DRI occurred between 16 and 24 DRI with 0.4%[-2.2-3.0]., Conclusion: For dlCT fat quantification in the presence of iron, material decomposition with combined reference values for iodine and iron delivers the most accurate results. Tube voltage-specific calibration of reference values is advisable while the impact of the DRI on dlCT fat quantification is neglectable., Competing Interests: The authors of this manuscript have the following competing interests: GM.C. is employed as a clinician scientist by Philips Health Care Germany. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors do not declare any competing interests. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Molwitz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)