Abstract Controlling gene expression is an instrumental tool for biotechnology, as it enables the dissection of gene function, affording precise spatial-temporal resolution. To generate this control, binary transactivational systems have been used employing a modular activator consisting of a DNA binding domain(s) fused to activation domain(s). For fly genetics, many binary transactivational systems have been exploited in vivo ; however as the study of complex problems often requires multiple systems that can be used in parallel, there is a need to identify additional bipartite genetic systems. To expand this molecular genetic toolbox, we tested multiple bacterially-derived binary transactivational systems in Drosophila melanogaster including the p -CymR operon from Pseudomonas putida , PipR operon from Streptomyces coelicolor , TtgR operon from Pseudomonas putida , and the VanR operon from Caulobacter crescentus . Our work provides the first characterization of these systems in an animal model in vivo . For each system we demonstrate robust tissue-specific spatial transactivation of reporter gene expression, enabling future studies to exploit these transactivational systems for molecular genetic studies.