We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.