Glucose homeostasis is a tightly coordinated process that ensures an adequate source of cellular energy. Blood glucose concentrations are coupled to changes in activity of glucokinase (GCK) in β-cells of the pancreas. GCK acts as the glucose sensor and is responsible for the phosphorylation of glucose that leads to insulin secretion from the pancreas to constrain glucose concentrations within physiologic levels. However, the underlying mechanistic details explaining how GCK maintains this tight control over glucose metabolism are unclear. First we elucidated the role of intracellular Ca2+, particularly Ca2+ mobilized from the endoplasmic reticulum, in GCK activation. We then investigated the cellular regulation of GCK through post-translational S-nitrosylation. Finally, we assessed changes in GCK activity that occur during the development of diabetes. To quantitatively assess this GCK activity in vitro, we used Förster resonance energy transfer (FRET) spectroscopy, fluorescence microscopy and GCK biosensors expressed in cultured pancreatic β-cells and primary mouse islets. This work describes our homotransfer FRET-GCK biosensor and improved data analysis methodology to understand the cellular regulation of GCK activity. To create the homotransfer biosensor, we attached two mVenus fluorescent proteins to GCK and transfected this single-color biosensor into cultured β-cells. We used the inherent polarization of light in combination with FRET principles to precisely measure GCK activation in living cells under various conditions.