Current methods to detect protein-protein interactions are either laborious to implement or not adaptable for mammalian systems or in vitro methods. By adding a peroxisomal targeting signal (PTS) onto one protein, binding partners lacking a targeting signal were co-transported into the peroxisomes in a "piggy-back" fashion, as visualized by confocal and electron microscopy. A fragment of colicin E2 and its tightly interacting immunity protein, ImmE2, were both expressed in the cytosol. When either one contained a PTS tag, both proteins were co-localized in the peroxisomes. The cytokine-independent survival kinase (CISK) containing a PTS tag was not efficiently targeted to the peroxisomes unless the Phox homology (PX) domain, attaching the protein to endosomal membranes, was removed. However, PTS-tagged CISK with deleted PX domain was able to direct 3-phosphoinositide-dependent protein kinase-1 (PDK-1) into the peroxisomes. This demonstrates that the two proteins interact in vivo. Mutating Ser486, which is phosphorylated in activated CISK, to Ala prevented the interaction, indicating that CISK and PDK-1 interact in a phosphorylation-dependent manner. The method therefore allows assessment of protein-protein interactions that depend on post-translational modifications that are cell-specific or dependent on the physiological state of the cell.