Gene expression biomarkers are now available for application in the identification of genotoxic hazards. The TGx‐DDI transcriptomic biomarker can accurately distinguish DNA damage‐inducing (DDI) from non‐DDI exposures based on changes in the expression of 64 biomarker genes. The 64 genes were previously derived from whole transcriptome DNA microarray profiles of 28 reference agents (14 DDI and 14 non‐DDI) after 4 h treatments of TK6 human lymphoblastoid cells. To broaden the applicability of TGx‐DDI, we tested the biomarker using quantitative RT‐PCR (qPCR), which is accessible to most molecular biology laboratories. First, we selectively profiled the expression of the 64 biomarker genes using TaqMan qPCR assays in 96‐well arrays after exposing TK6 cells to the 28 reference agents for 4 h. To evaluate the classification capability of the qPCR profiles, we used the reference qPCR signature to classify 24 external validation chemicals using two different methods—a combination of three statistical analyses and an alternative, the Running Fisher test. The qPCR results for the reference set were comparable to the original microarray biomarker; 27 of the 28 reference agents (96%) were accurately classified. Moreover, the two classification approaches supported the conservation of TGx‐DDI classification capability using qPCR; the combination of the two approaches accurately classified 21 of the 24 external validation chemicals, demonstrating 100% sensitivity, 81% specificity, and 91% balanced accuracy. This study demonstrates that qPCR can be used when applying the TGx‐DDI biomarker and will improve the accessibility of TGx‐DDI for genotoxicity screening. Environ. Mol. Mutagen. 60: 122–133, 2019. © 2018 Her Majesty the Queen in Right of Canada Environmental and Molecular Mutagenesis.