Ethical considerations surrounding the utilization of animals in scientific research have prompted a widespread search for alternative methodologies. This review explores the historical context and ethical dilemmas associated with traditional animal testing methods, before introducing the Monocyte Activation Test (MAT) as a promising alternative, and outlining its basic principles, historical development, and advantages over conventional animal testing. The role of monocytes in the immune system and the activation pathways utilized in MAT are elucidated, while regulatory acceptance and guidelines for MAT validation are introduced, alongside case studies proving its reliability and reproducibility. The applications of MAT in pharmaceutical and medical device testing are summarized, together with its potential future uses. Although the MAT faces limitations and challenges, the global perspective to reduce unnecessary animal tests has become a general concept in animal welfare and scientific research.