The reliance on animal models in biomedical research and education has long been considered essential, yet ethical concerns, translational limitations, and evolving regulatory frameworks are driving a shift toward innovative non-animal alternatives. This chapter examines the emergence of organoids and organ-on-chip technologies—advanced three-dimensional (3D) systems that are used to simulate human tissue architecture and function. These systems enable researchers to model disease mechanisms and therapeutic responses in vitro, offering dynamic, human-relevant environments that replicate physiological and pathological conditions. Organoids, often derived from patient-derived stem cells, support personalized medicine, while organ-on-chip systems incorporate microfluidics to mimic complex biological processes such as blood flow and mechanical stress. Together with advances in micro-bioengineering and 3D printing, these technologies are reducing reliance on animal testing, and align with the principles of the 4Rs—Reduction, Refinement, Replacement, and Responsibility. These systems have the potential to significantly improve disease modelling, drug development, diagnostics, and biomedical education.