Bimolecular fluorescence complementation (BiFC) assay is an innovative approach to investigate protein interactions, based on the reassembly of protein fragments of the fluorescent proteins which directly report interactions. The fluorescent proteins tolerate circular permutation and insertions of foreign proteins with maintenance of fluorescence. So when the proteins fused to the reporter fragments interact with each other, a direct readout of the association would be given from the facilitating reassembly of the active reporter protein. Moreover, with distinct spectra difference of the fluorescent protein family members, BiFC assay is expanded to multicolor BiFC assay which enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners. From it first reported in Molecular Cell in 2002 till now, this approach has been used on the networks of protein interaction in mammal cells, plant cells or even E.coli, and researches on transcription factors, G protein βγ dimmers, protein ubiqutination and so on.