Diet can affect a spectrum of biological processes ranging from behavior to cellular metabolism. Yet, the precise role of an individual dietary constituent can be a difficult variable to isolate experimentally. A chemically defined food (CDF) permits the systematic evaluation of individual macro- and micronutrients. In addition, CDF facilitates the direct comparison of data obtained independently from different laboratories. Here, we report the development and characterization of a CDF for Drosophila. We show that CDF can support the long-term culture of laboratory strains and demonstrate that this formulation has utility in isolating macronutrient from caloric density requirements in studies of development, longevity and reproduction.