Recently, numerous organizations, including governmental regulatory agencies in the U.S. and abroad, have proposed using data from New Approach Methodologies (NAMs) for augmenting and increasing the pace of chemical assessments. NAMs are broadly defined as any technology, methodology, approach or combination thereof that can be used to provide information on chemical hazard and risk assessment that avoids the use of intact animals. High-throughput transcriptomics (HTTr) is a type of NAM that uses gene expression profiling as an endpoint for rapidly evaluating the effects of large numbers of chemicals on in vitro cell culture systems. As compared to targeted high-throughput screening (HTS) approaches that measure the effect of chemical X on target Y, HTTr is a non-targeted approach that allows researchers to more broadly characterize the integrated response of an intact biological system to chemicals that may affect a specific biological target or many biological targets under a defined set of treatment conditions (time, concentration, etc.). HTTr screening performed in concentration-response mode can provide potency estimates for the concentrations of chemicals that produce perturbations in cellular response pathways. Here, we discuss study design considerations for HTTr concentration-response screening and present a framework for the use of HTTr-based biological pathway-altering concentrations (BPACs) in a screening-level, risk-based chemical prioritization approach. The framework involves concentration-response modeling of HTTr data, mapping gene level responses to biological pathways, determination of BPACs, in vitro-to-in vivo extrapolation (IVIVE) and comparison to human exposure predictions.