Two-component signaling is the primary means by which bacteria, archaea and certain eukaryotes sense and respond to their environments. Signal transfer proceeds through sequential His-to-Asp phosphorylation of upstream histidine kinases and downstream response regulators. These systems share highly modular designs and have been incorporated into a myriad of cellular processes. The highly labile chemical natures of phosphoaspartate and phosphohistidine lead to relatively short experimental life-times, making study of the modified signaling proteins challenging. The focus of this research was to develop computational and experimental approaches for characterizing phosphorylated two-component signaling proteins. Following an introductory chapter, the first experimental section presents a computational technique for simulating the activation of individual response regulator proteins. This is accomplished using known experimental data on conserved active site chemistry to define a common set of restraints to drive each simulation. The protocol was verified on five genetically diverse response regulators with known experimental structures. The second section applies this principle to signaling complexes to study the effects of phosphorylation on protein- protein interactions within the Saccharomyces cerevisiae osmoregulatory signaling system. The third section describes the experimental characterization of a specific signaling complex from Saccharomyces cerevisiae between the response regulator Ssk1 and a point mutant (G68Q) of the histidine phosphotransfer protein Ypd1 using X-ray crystallography. This mutation occurs near the active site of both proteins and appears to interfere with phosphotransfer. Further in silico studies were performed to observe the role of G68 in catalysis of phosphotransfer.