This protocol is designed to prepare adult axenic Drosophila before monitoring their behavior in a two-choice feeding assay, where flies are confronted with an axenic versus a dead or alive bacteria-contaminated feeding solution. Several aspects of the procedure, including raising and aging flies in axenic conditions, starving adult flies, and composing feeding solutions, are detailed. The bacterium used in this protocol, Erwinia carotovora carotovora-15 2141 (Ecc-15 2141 ), is commonly used to decipher the mechanisms controlling host-pathogen interactions in the Drosophila model. For complete details on the use and execution of this protocol, please refer to Charroux et al. (2020).