Histidine kinases are one of the two proteins that constitute a two-component signal transduction system. These systems are used by bacteria, fungi, and several plants to sense the conditions of their environment and alter their behavior to ensure survival. In bacteria, two-component systems control basic cellular processes like motility, virulence, cell division, sporulation etc. While histidine kinases are responsible for signal perception in the host, the other protein of this system, called response regulator, conducts the response output. These response outputs can be downstream gene regulation via DNA binding, RNA binding, enzymatic reactions etc. Although a significant number of histidine kinases have been identified for Clostridioides difficile till date, it is not yet clear how these histidine kinases affect the pathogenicity of this bacterium, especially sporulation, a process where bacterial cell goes into a dormant form until placed in a favorable environment. The precise prediction of progression mechanism of sporulation adopted by this bacterium could enable researchers to develop techniques to mitigate this pathogenic behavior. In general, these two-component systems (TCS) are absent in mammals and hence can be a good target for therapeutics for several bacterial and fungal infections. This thesis presents a detailed script of studies done towards unraveling the mechanism behind sporulation of Clostridioides difficile. This includes using γ-32P ATP functional assay to provide quantitative and qualitative evidence of functional activities of sporulation-related histidine kinases as well as initial binding screens to examine the impetus behind these phosphorelay systems. Histidine kinases covered in this study are HK_1587 from the hypervirulent R20291 strain and CD1492 and CD2492 from the historic CD630 strain of C. difficile.