The increasing number of compounds under development and chemicals in commerce that require safety assessments pose a serious challenge for regulatory agencies worldwide. In vitro screening using toxicogenomic biomarkers has been proposed as a first-tier screen in chemical assessment and has been endorsed internationally. We previously developed, evaluated, and validated an in vitro transcriptomic biomarker responsive to DNA damage-inducing (DDI) agents, namely TGx-DDI, for genotoxicity testing in human cells and demonstrated the feasibility of using TGx-DDI in a medium-throughput, cell-based genotoxicity testing system by implementing this biomarker with the Nanostring nCounter system. In this current study, we took advantage of Nanostring nCounter Plexset technology to develop a highly auto­mated, multiplexed, and high-throughput genotoxicity testing assay, designated the TGx-DDI Plexset assay, which can increase the screening efficiency eight-fold compared to standard nCounter technology while decreasing the hands-on time. We demonstrate the high-throughput capability of this assay by eliminating concentration determination and RNA extraction steps without compromising the specificity and sensitivity of TGx-DDI. Thus, we propose that this simple, highly automated, multiplexed high-throughput pipeline can be widely used in chemical screening and assessment.