Recently we reported a novel protein-protein interaction assay FlimPIA (firefly luminescent intermediate-based protein-protein interaction assay) based on the functional complementation of two mutant firefly luciferases (Fluc), each defective in its one of two half reactions. The assay detects approximation of two mutant Flucs, namely, a "Donor" that catalyzes ATP-driven luciferin adenylation to produce a luciferyl-adenylate intermediate, and an "Acceptor" that mainly catalyzes subsequent oxidative luminescent reaction. However, there was a problem in FlimPIA that the remaining adenylation activity of the Acceptor constituted its background signal and hampered its wider use. In this study, we aimed at reducing the background signal by trapping the Acceptor to the "oxidation" conformation, either chemically or by disulfide bonding. The results showed higher sensitivity and detection over the longer distance of the developed assay compared to conventional FlimPIA, Fluc-based protein-fragment complementation assay, and fluorescent protein-based FRET assay.