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Research using experimental animals has substantially contributed to advances in science and medicine. Animal experiments are nearly essential for biomedical research and development efforts. Because many animals are sacrificed, researchers should consider the welfare of experimental animals and related ethical issues, along with the successful results of their experiments. This review introduces the criteria that should be considered in terms of experimental animal ethics, based on the principles of the 3 R’s: replacement, representing careful consideration of the need for animal experiments; reduction, representing the use of the minimal number of animals to obtain meaningful experimental results; and refinement, representing continuous effects to find alternative methods to reduce pain and distress in experimental animals. Based on these principles, the following points should be considered when planning experiments: the necessity of animal experiments; alternatives to animal experiments; the relevance of the species and numbers of experimental animals; appropriate assessment and management of pain; the proper usage of sedatives, painkillers, and anesthesia; and valid timing for humane endpoints and euthanasia. These criteria are beneficial for both experimental animals and researchers because careful handling to ensure experimental animal welfare guarantees that scientific research will yield convincing, repeatable, and accurate results.