Artificially reared larvae are an ideal model for experiments involving brood diseases or testing pesticides. Larvae development condition can influence the general performance of adult honey bees but little is known about queen larvae. We first performed a review of existing protocols. Based on existing protocols we tested and changed parameters linked to breeding conditions. The aims of this study were to compare and determine the best protocol for in vitro queen larvae rearing. For this purpose, we tested 57 sub-protocols both available in the literature and new protocols to compare their effectiveness and to identify potential improvement. As a major result of this study we found low queen emerging rate in most of the sub-protocols. The size and the type of rearing cup, diet quantity (food can be done ad-libitum but a lower volume (i.e., 270 mu l) is also highly suitable) and quality including freshness of the royal jelly among other variables contribute to the conjunction of numerous parameters that makes queen in vitro rearing complex. The high number of rearing conditions tested here facilitates emerging rate comparison. The available literature and the current study highlight the complexity of searching for the best protocol and necessitate further investigation to provide a suitable protocol for in vitro queen rearing. Such protocol will be very needed to facilitate larvae development of future queens and evaluate the consequences of other stressors (parasites, pollutants ...) in control condition on queen health.