The evaluation of nitrosamine drug substance-related impurities (NDSRIs) has become a regulatory priority due to potential carcinogenicity. Previously, we evaluated mutagenicity and genotoxicity of NDSRIs using the enhanced Ames Test (EAT) and human TK6 cells. In this study, we investigated the genotoxicity of ten of these NDSRIs using metabolically competent human HepaRG cells. DNA damage and micronucleus (MN) formation were evaluated in both 2D and 3D models using the CometChip and flow-cytometry-based MN assays, respectively. After 24-h exposure, five EAT-positive NDSRIs, N-nitroso-duloxetine, N-nitroso-fluoxetine, N-nitroso-lorcaserin, N-nitroso-nortriptyline, and N-nitroso-varenicline, significantly induced DNA damage in both 2D and 3D models and increased MN and γH2A.X formation in 3D spheroids. Only three EAT-positive NDSRIs, N-nitroso-duloxetine, N-nitroso-fluoxetine, and N-nitroso-nortriptyline, increased MN frequency in 2D cultures. The five EAT-negative NDSRIs, N-nitroso-diclofenac, N-nitroso-folic acid, N-nitroso-paroxetine, N-nitroso-desvaleryl-valsartan, and N-nitroso-desvaleryl-valsartan methyl ester, showed no DNA damage or MN formation in either model. Quantitative comparisons showed that N-nitroso-nortriptyline was the most potent genotoxicant in HepaRG cells. Overall, the ten NDSRIs exhibited the same positive/negative genotoxicity outcomes in both the EAT and 3D HepaRG spheroids. These findings support the use of 3D HepaRG spheroids as an alternative in vitro model for detecting NDSRI-induced genotoxicity and confirming NDSRI responses in the EAT.