N-Nitrosamines (NAs) are probable human carcinogens and were detected as impurities in pharmaceuticals, which led to a concern for human health. NAs require metabolic activation before they become mutagenic, and not all NAs are mutagenic since their reactivity is related to their structure. While some NAs are potent mutagens in vivo, in vitro metabolization with exogenous S9 liver extract is generally less efficient. While an enhanced bacterial mutagenicity protocol was recently developed, which uses increased concentrations of S9 liver extracts, there presently is not an improved metabolization protocol suitable for mammalian cell genotoxicity assays. Therefore, we optimized a hamster S9 liver extract-based protocol for in vitro NA metabolization and assessed the genotoxic potential of various NAs using ToxTracker. With this enhanced metabolization protocol (EMP), the genotoxic potency of N-nitrosodimethylamine (NDMA) increased approximately 200-fold compared with the standard S9 liver extract-based exposure protocol in ToxTracker. The EMP was further validated with seven additional mutagenic NAs to which humans are commonly exposed: N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDELA), N-nitrosodibutylamine (NDBA), N-nitrosofluoxetine (NF), 1-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), and 1-cyclopentyl-4-nitrosopiperazine (CPNP), and two non-mutagenic NAs: N-nitrosobupropion (NBuPRO) and N-nitrosoproline (NPRO). Genotoxicity could be confirmed for six NAs using the EMP, demonstrating that mammalian cells and the new approach methodology (NAM) ToxTracker may have potential when investigating NA-related genotoxicity.