Animal experimentation has played a crucial role in scientific and medical advancements, yet it remains a subject of ethical debate. Concerns primarily revolve around animal welfare, the moral justification for their use, and the distress they may experience during experiments. The ethical dilemma of using research animals becomes more difficult when placed in the context of doubts about the ability to extrapolate results from animals to humans. To address this, Ethics Committees have adopted the “4R” principles: Reduction, Refinement, Replacement, and Responsibility to balance scientific requirements with animal welfare. Although alternative technologies such as “organ-on-a-chip,” 3D culture, and computational modeling open up new prospects, they are still unable to fully simulate the complexity of living organisms, and the full implementation of this principle continues to face many barriers. Furthermore, strict regulations and limited financial resources continue to make animal models central to the validation and approval of research. In some cases, animal research is still conducted to meet review criteria, even for traditional medicines that are safe in humans. Therefore, there is a need to raise awareness among funding and regulatory agencies to reduce unnecessary animal use. Despite the rapid development of non-animal research, the complete elimination of animal models remains a challenge. This chapter examines the role and ethical debates surrounding animal testing, the limits of current alternatives, and the systemic barriers that impede translation. It proposes an approach to reconciling scientific progress and ethical values in biomedical research.