Bacterial signaling systems regulate vast physiological functions, from general stress responses to consumption of nutrients. Their study is often limited to the interrogation of single systems within model bacterial species, such as Escherichia coli. This framework is limiting, because it does not consider evolutionary relationships between the hundreds of systems on a single chromosome and omits the context of other extant non-model bacterial species. In this thesis, I make a case for a holistic approach to interrogate the functions of bacterial signaling systems. I develop a suite of high-throughput experimental and computational methods that when implemented elucidate the functional and evolutionary relationships between bacterial two-component systems. A holistic view of bacterial signaling systems might one day be utilized as a general tool for functional annotation and has the potential to unlock networks of regulation for non-model bacterial species.