Polyclonal antibodies, widely used in research and diagnostics, are conventionally isolated from the blood of immunised mammals. The fact that antibodies can also be detected in the yolk of eggs laid by immunised hens, led to the development of the yolk antibody technology as an alternative method less stressful to animals. Hens can be kept under nearly natural conditions and antibodies be isolated from the eggs. Further, the IgY-technology has the advantage that avian antibodies can be produced against several antigens which do not induce a immune response in mammals. Protocols on how to keep and immunise hens and on the extraction, isolation and use of antibodies from yolk are described in detail in this manual. For their work on yolk antibodies the authors received the FISEA (International Foundation for the Substitution of Animal Experimentation, Luxembourg) prize in 1997.