ABSTRACT Gene mutations can be detected in mammalian cells in vitro using indicator genes such as the hypoxanthine‐guanine‐phosphoribosyltransferase (HPRT) gene. These assays have been adopted as OECD test guidelines (TG, e.g., OECD TG no. 476) and are used for regulatory purposes. The in vitro transgenic rodent assay (TGRA) in primary MutaMouse hepatocytes is a novel approach for the detection and quantification of gene mutations. Its methodology follows the same principles as the in vivo TGRA, an in vivo gene mutation assay with regulatory adoption (OECD TG no. 488). Although the potential of the in vitro TGRA to identify mutagens has been reported, its performance compared to an established in vitro gene mutation assay has not been reported. This study compared the in vitro TGRA with the HPRT assay using 10 known in vivo mutagens. The in vitro TGRA correctly identified all 10 mutagens, whereas the HPRT assay identified only nine. Benchmark concentration (BMC) modeling for the nine substances detected by both assays revealed overlapping confidence intervals for six compounds, indicating comparable sensitivity. For three mutagens, the HPRT assay yielded lower BMC intervals. Additionally, eight substances known to be non‐mutagenic in vivo tested negative in the in vitro TGRA. While increased cytotoxicity did not induce increased mutant frequencies, it reduced DNA yield, thereby impairing mutagenicity assessment. The results of this study contribute to the understanding of the sensitivity and robustness of the in vitro TGRA and provide essential information for the validation of the assay.