Recent genetic and clinical evidence has implicated glucokinase regulatory protein (GKRP) in the pathogenesis of type 2 diabetes and related traits. The primary role of GKRP is to bind and inhibit hepatic glucokinase (GCK), a critically important protein in human health and disease that exerts a significant degree of control over glucose metabolism. As activation of GCK has been associated with improved glucose tolerance, perturbation of the GCK-GKRP interaction represents a potential therapeutic target for pharmacological modulation. Recent structural and kinetic advances are beginning to provide insight into the interaction of these two proteins. However, tools to comprehensively assess the GCK-GKRP interaction, particularly in the context of small molecules, would be a valuable resource. We therefore developed three robust and miniaturized assays for assessing the interaction between recombinant human GCK and GKRP: an HTRF assay, a diaphorase-coupled assay, and a luciferase-coupled assay. The assays are complementary, featuring distinct mechanisms of detection (luminescence, fluorescence, FRET). Two assays rely on GCK enzyme activity modulation by GKRP while the FRET-based assay measures the GCK-GKRP protein-protein interaction independent of GCK enzymatic substrates and activity. All three assays are scalable to low volumes in 1536-well plate format, with robust Z' factors (>0.7). Finally, as GKRP sequesters GCK in the hepatocyte nucleus at low glucose concentrations, we explored cellular models of GCK localization and translocation. Previous findings from freshly isolated rat hepatocytes were confirmed in cryopreserved rat hepatocytes, and we further extended this study to cryopreserved human hepatocytes. Consistent with previous reports, there were several key differences between the rat and human systems, with our results suggesting that human hepatocytes can be used to interrogate GCK translocation in response to small molecules. The assay panel developed here should help direct future investigation of the GCK-GKRP interaction in these or other physiologically relevant human systems.