Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable weak PPI detection in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL’s ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a new class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL’s ability to expedite PPI analysis, assess target specificity and cell permeability, and to reveal off-target effects of PPI modifiers should facilitate development of effective, cell permeable PPI therapeutics and elaboration of diverse biological mechanisms.