Significance Standard in vitro assays to assess genotoxicity frequently generate positive results that are subsequently found to be irrelevant for in vivo carcinogenesis and human cancer risk assessment. Currently used follow-up methods, such as animal testing, are expensive and time-consuming, and the development of approaches enabling more accurate mechanism-based risk assessment is essential. We developed an in vitro transcriptomic biomarker-based approach that provides a robust biomarker reflecting stress-signaling responses. The biomarker correctly identifies the vast majority of irrelevant genotoxicity results from in vitro chromosome damage assays. TGx-DDI, a multigene biomarker for DNA damage-inducing agents, is the first biomarker that not only shows convincing interlaboratory and intralaboratory reproducibility, but also performs accurately in a system suitable for high-throughput screening.