Although many decades of research have been conducted on bacterial sensor kinases, their mechanism of transmembrane signal transduction remains unclear. Here, we have compiled a set of methodologies that we believe would be beneficial to any research group starting studies on their sensor kinase of interest. Alternatively, these methods would be beneficial to those monitoring evolution of sensor kinases or potentially to those engineering sensor kinases for a particular function. To accomplish this, we began by comparing the previously determined orientation of the transmembrane domain (TMD) in native bacterial membranes to a structure generated in silico after atomistic molecular dynamics (MD) simulation. This comparison showed that the in silico structure was an accurate representation of the EnvZ TMD in native bacterial membranes. Due to this similarity, we mapped previously existing data that assessed the change in EnvZ signal output from a set of ~60 Cys substitutions onto the structure generated in silico and found that water is present near where the sensor kinase becomes activated by Cys mutations. For brevity, we solely focus on functional water encapsulation within the TMD of EnvZ, but other parameters such as individual helical position, tilt, or rotation and their role in signal transduction could also be assessed. We hope that other research groups find this workflow useful and decide to employ it to assess their sensor kinases of interest.