The bimolecular fluorescence complementation (BiFC) assay is a versatile technique for investigating protein-protein interaction (PPI) in living systems. The BiFC assay exploits the color-emitting moiety and the modular structure of fluorescent proteins to provide both temporal and spatial information of the PPI. The modular property of fluorescent proteins enables researchers to strategically partition a fluorescent protein into two nonfluorescent units, which can be independently fused to other proteins. When the fusion proteins interact with each other, the nonfluorescent fragments reconstitute to generate a fluorescence signal. PPI can then be detected by capturing the fluorescence signal with a fluorescence microscope. In this chapter, the Venus fluorescent protein is employed to demonstrate the application of the BiFC assay.