In response to the need to minimize the use of experimental animals, new approach methodologies (NAMs) using advanced technology have emerged in the 21st century. ToxCast/Tox21 aims to evaluate the adverse effects of chemicals quickly and efficiently using a high-throughput screening and to transform the paradigm of toxicity assessment into mechanism-based toxicity prediction. The ToxCast/Tox21 database, which contains extensive data from over 1400 assays with numerous biological targets and activity data for over 9000 chemicals, can be used for various purposes in the field of chemical prioritization and toxicity prediction. In this study, an overview of the database was explored to aid mechanism-based chemical prioritization and toxicity prediction. Implications for the utilization of the ToxCast/Tox21 database in chemical prioritization and toxicity prediction were derived. The research trends in ToxCast/Tox21 assay data were reviewed in the context of toxicity mechanism identification, chemical priority, environmental monitoring, assay development, and toxicity prediction. Finally, the potential applications and limitations of using ToxCast/Tox21 assay data in chemical risk assessment were discussed. The analysis of the toxicity mechanism-based assays of ToxCast/Tox21 will help in chemical prioritization and regulatory applications without the use of laboratory animals.