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Human settlements on the Moon, crewed missions to Mars and space tourism will become a reality in the next few decades. Human presence in space, especially for extended periods of time, will therefore steeply increase. However, despite more than 60 years of spaceflight, the mechanisms underlying the effects of the space environment on human physiology are still not fully understood. Animals, ranging in complexity from flies to monkeys, have played a pioneering role in understanding the (patho)physiological outcome of critical environmental factors in space, in particular altered gravity and cosmic radiation. The use of animals in biomedical research is increasingly being criticized because of ethical reasons and limited human relevance. Driven by the 3Rs concept, calling for replacement, reduction and refinement of animal experimentation, major efforts have been focused in the past decades on the development of alternative methods that fully bypass animal testing or so-called new approach methodologies. These new approach methodologies range from simple monolayer cultures of individual primary or stem cells all up to bioprinted 3D organoids and microfluidic chips that recapitulate the complex cellular architecture of organs. Other approaches applied in life sciences in space research contribute to the reduction of animal experimentation. These include methods to mimic space conditions on Earth, such as microgravity and radiation simulators, as well as tools to support the processing, analysis or application of testing results obtained in life sciences in space research, including systems biology, live-cell, high-content and real-time analysis, high-throughput analysis, artificial intelligence and digital twins. The present paper provides an in-depth overview of such methods to replace or reduce animal testing in life sciences in space research.