Originally, a method to rear worker honey bee larvae in vitro was introduced into the field of bee biology to analyse honey bee physiology and caste development. Recently, it has become an increasingly important method in bee pathology and toxicology. The in vitro method of rearing larvae is complex and can be developed as an art by itself, especially if the aim is to obtain queens or worker bees which, for example, can be re-introduced into the colony as able members. However, a more pragmatic approach to in vitro rearing of larvae is also possible and justified if the aim is to focus on certain pathogens or compounds to be tested. It is up to the researcher(s) to decide on the appropriate experimental establishment and design. This paper will help with this decision and provide guidelines on how to adjust the method of in vitro rearing according to the specific needs of the scientific project.