Traditionally, safety evaluation at the early stage of drug discovery research has been done using in silico, in vitro, and in vivo systems in this order because of limitations on the amount of compounds available and the throughput ability of the assay systems. While these in vitro assays are very effective tools for detecting particular tissue-specific toxicity phenotypes, it is difficult to detect toxicity based on complex mechanisms involving multiple organs and tissues. Therefore, the development of novel high throughput in vivo evaluation systems has been expected for a long time. The zebrafish (Danio rerio) is a vertebrate with many attractive characteristics for use in drug discovery, such as a small size, transparency, gene and protein similarity with mammals (80% or more), and ease of genetic modification to establish human disease models. Actually, in recent years, the zebrafish has attracted interest as a novel experimental animal. In this article, the author summarized the features of zebrafish that make it a suitable laboratory animal, and introduced and discussed the applications of zebrafish to preclinical toxicity testing, including evaluations of teratogenicity, hepatotoxicity, and nephrotoxicity based on morphological findings, evaluation of cardiotoxicity using functional endpoints, and assessment of seizure and drug abuse liability.