Superpositive green fluorescent protein (GFP) is a designed recombinant bimolecular form of sg100 GFP, the most commonly used variation of native GFP, with a high theoretical net charge, which reduces aggregation, achieved by replacement of aspartic acid residues with arginine and lysine residues. Here, we expressed superpositive GFP as separate N-terminal and C-terminal domains (split superpositive GFP) in order to develop an assay for the detection of proteins binding to one another and reconstituting functional GFP fluorescence.This recovery of fluorescence upon protein pair binding provides an easily observed and semi-quantitatively measurable analogue for the successful interaction of the proteins. The objective of this project was to use the split-superpositive GFP protein complementation assay in conjunction with free or orthogonally reporting inhibitor proteins. We hoped to find that the presence of high affinity free inhibitor proteins would correlate with a decrease in recovered fluorescence when compared to cells in which a lower affinity inhibitor, or no inhibitor, is present. As a secondary objective, we also tested whether an inhibitor conjugated to the N terminal fragment of split cerulean fluorescent protein would be capable of providing an orthogonal cerulean signal upon competitive binding with the C terminal fragment of split-superpositive GFP. Evaluating time dependent evolution of fluorescence within cells expressing this system using flow cytometry could provide a novel method of screening macromolecular protein-protein interactions and inhibitors of these interactions rapidly and easily.