High costs, long test times, and societal concerns related to animal use have required the development of in vitro assays for the rapid and cost-effective toxicological evaluation and characterization of compounds in both the pharmaceutical and environmental arenas. Although the pharmaceutical industry has developed very effective, high-throughput in vitro assays for determining the therapeutic potential of compounds, the application of this approach to toxicological screening has been limited. A primary reason for this is that while drug candidate screens are directed to a specific target/mechanism, xenobiotics can cause toxicity through any of a myriad of undefined interactions with cellular components and processes. Given that it is not practical to design assays that can interrogate each potential toxicological target, an integrative approach is required if there is to be a rapid and low-cost toxicological evaluation of chemicals. Cellular stress response pathways offer a viable solution to the creation of a set of integrative assays as there is a limited and hence manageable set (a small ensemble of 10 or less) of major cellular stress response pathways through which cells mount a homoeostatic response to toxicants and which also participate in cell fate/death decisions. Further, over the past decades, these pathways have been well characterized at a molecular level thereby enabling the development of high-throughput cell-based assays using the components of the pathways. Utilization of the set of cellular stress response pathway-based assays as indicators of toxic interactions of chemicals with basic cellular machinery will potentially permit the clustering of chemicals based on biological response profiles of common mode of action (MOA) and also the inference of the specific MOA of a toxicant. This article reviews the biochemical characteristics of the stress response pathways, their common architecture that enables rapid activation during stress, their participation in cell fate decisions, the essential nature of these pathways to the organism, and the biochemical basis of their cross-talk that permits an assay ensemble screening approach. Subsequent sections describe how the stress pathway ensemble assay approach could be applied to screening potentially toxic compounds and discuss how this approach may be used to derive toxicant MOA from the biological activity profiles that the ensemble strategy provides. The article concludes with a review of the application of the stress assay concept to noninvasive in vivo assessments of chemical toxicants.