Gene mutations can be detected in mammalian cells in vitro using indicator genes like 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), typically performed in primary MutaMouse hepatocytes, is a novel approach for the detection and quantification of gene mutations. Its performance resembles the in vivo TGRA, an in vivo gene mutation assay that has been adopted as OECD TG (no. 488). Although the potential of the in vitro TGRA to identify mutagens has been reported, the performance of this assay as 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 ten known in vivo mutagens. The in vitro TGRA correctly identified all ten mutagens, whereas the HPRT assay identified only nine. Benchmark concentration (BMC) modelling 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 were tested in the in vitro TGRA, where applicable up to cytotoxic concentrations. While cytotoxicity did not induce increased mutant frequencies, it reduced DNA yield, impairing mutagenicity assessment. The results of this study add to the information on the sensitivity and robustness of the in vitro TGRA and provide essential information for the validation of the assay.