The purpose of our study is to determine a threshold for iodine quantification to distinguish definitely non-enhancing benign renal lesions from potential enhancing masses on portal venous dual-layer spectral computed tomography (CT) to reduce the need for additional multiphase CT. In this single-center retrospective study, patients (≥18 years) scanned between April 2021 and January 2023 following the local renal CT protocol were included. Exclusion criteria were patients without renal lesions, lesions smaller than 10 mm, only fat-containing lesions, abscesses or infarction, follow-up after radiofrequent ablation, wrong scan protocol, or artefacts. Scans were performed on a dual layer detector-based spectral CT (CT 7500, Philips Healthcare, Best, The Netherlands). Iodine concentration (mgI/mL) in renal lesions was determined using spectral data. Analyses were performed for all lesions and for lesions of >30 HU on portal venous CT. Enhancement on multiphase CT (≥20 ΔHU from true unenhanced (TUE) to portal venous phase (PVP) CT) was used as reference standard. To determine thresholds for iodine concentration receiver operating characteristic (ROC) curves, area under the curve (AUC) and 95% confidence intervals were calculated. To obtain thresholds for definite (non-)enhancement, 100% sensitivity with maximum specificity and 100% specificity with maximum sensitivity were noted. Data were measured using one reader. To assess interobserver agreement, a second reader performed measurements on the PVP CT scans. A total of 103 patients (62 years ± 14, 68 men) were included. We measured 328 renal lesions, 56 enhancing lesions (17%) in 38 patients and 272 non-enhancing lesions (83%) in 86 patients. The threshold for non-enhancing lesions was 0.76 mgI/mL or lower (100% sensitivity, 76% specificity). The threshold for a definite enhancing mass was 1.69 mgI/mL or higher (100% specificity, 78% sensitivity). A total of 77% of indeterminate lesions (>30 HU on PVP CT) in our study could be definitely characterized. Renal lesions can be definitively classified as non-enhancing or enhancing on PVP spectral CT using thresholds of 0.76 mgI/mL or 1.69 mgI/mL, respectively, eliminating the need for multiphase imaging.