As several diseases have been linked to dysbiosis of the human intestinal microflora, manipulation of the microbiota has emerged as an exciting new strategy for potentially treating and preventing diseases. However, the human microbiota consists of a plethora of different species, and distinguishing the impact of a specific bacterial species on human health is challenging. In tackling this challenge, the fruit fly Drosophila melanogaster, with its far simpler microbial composition, has emerged as a powerful model for unraveling host-microbe interactions. To study the interplay between the resident commensal microbiome and the host, flies can be made germ-free, or axenic. To elucidate the impact of specific bacteria, axenic flies can then be re-introduced to specific microbial species. In this unit, we provide a step-by-step protocol on how to rear Drosophila melanogaster under axenic conditions and confirm the axenity of flies.