BACKGROUND: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays. OBJECTIVES: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function. METHODS: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans. RESULTS: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization. CONCLUSIONS: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. https://doi.org/10.1289/EHP2589.