"Replacement" is one of the alternatives to animals in research know as the "3R's" as described in The Principles of Humane Experimental Technique. Authors Russell and Burch define a replacement technique as "any scientific method employing non-sentient material which may in the history of experimentation replace methods which use conscious living vertebrates." Scientists should seek to replace animals in experiments with alternatives whenever possible.

Replacement alternatives include the use of human volunteers, in vitro systems, mathematical and computer models and simulations, microorganisms, bacteria, plants and fungi, and imaging techniques. Some replacements can be done completely without use of other living beings - computer models and simulations for example. At present, however, there is a far greater reliance on in vitro replacements that rely on cells or tissues taken from donor animals, as well as from humans and early-development human or animal embryos. A single donor may provide tissue that can be used for a large number of cultures and alleviate the need for multiple test subjects.

Partial replacement is also possible. For example, alternative methods might be used to pre-screen substances for toxicity. If preliminary in vitro tests show the substance is toxic, there is no need to test on animals. Only a negative result would lead to additional testing on animals.

A thorough review of the scientific literature can help researchers find replacements - as it may indicate that the work has been done previously or that there are other scientifically valid means to obtain the desired data.