p53 plays an important role in regulating a wide variety of cellular processes, such as cell cycle arrest and/or apoptosis. Dysfunction of p53 is frequently associated with several pathologies, such as cancer and neurodegenerative diseases. In recent years substantial progress has been made in developing novel p53-activating molecules. Importantly, modulation of p53 interaction with its main inhibitor, Mdm2, has been highlighted as a promising therapeutic target. In this regard, bimolecular fluorescence complementation (BiFC) analysis, by providing direct visualization of protein interactions in living cells, offers a straightforward method to identify potential modulators of protein interactions. In this study, we developed a simple and robust Venus-based BiFC system to screen for modulators of p53–p53 and p53–Mdm2 interactions in live mammalian cells. We used nutlin-3, a well-known disruptor of p53–Mdm2 interaction, to validate the specificity of the assay. The reduction of BiFC signal mediated by nutlin-3 was correlated with an increase in Puma transactivation, PARP cleavage, and cell death. Finally, this novel BiFC approach was exploited to identify potential modulators of p53–Mdm2 complex formation among a commercially available chemical library of 33 protein phosphatase inhibitors. Our results constitute “proof-of-concept” that this model has strong potential as an alternative to traditional target-based drug discovery strategies. Identification of new modulators of p53–p53 and p53–Mdm2 interactions will be useful to achieve synergistic drug efficacy with currently used anti-tumor therapies.