Protein-Protein Interactions (PPIs) play essential roles in diverse biological processes and their misregulations are associated with a wide range of diseases. Especially, the growing attention on PPIs as a new class of therapeutic target is increasing the need for an efficient way of cell-based PPI analysis. Thus, we newly developed a robust PPI assay (SeePPI) based on the co-translocation of interacting proteins to the discrete subcellular compartment ‘processing body’ (p-body) inside living cells, enabling a facile analysis of PPI by the enriched fluorescent signal. The feasibility and strength of SeePPI, Signal enhancement exclusively on P-body for Protein-protein Interaction, assay was firmly demonstrated with FKBP12/FRB interaction instantly induced by rapamycin within seconds in real-time analysis of living cells, indicating its recapitulation of physiological PPI dynamics. In addition, we applied p53/MDM2 interaction and its dissociation by Nutlin-3 into SeePPI assay and further confirmed that SeePPI was quantitative and well reflected the endogenous PPI. Our SeePPI assay will provide another useful tool to achieve an efficient analysis of PPIs and their modulators in cells.