One way that cells control the flow of signaling information is with protein interaction domains. One such domain, the PDZ domain, is responsible for a wide range of cellular activities, from protein scaffolding to synaptic plasticity, and has been implicated in the pathology of multiple diseases. The >300 PDZ domains in the human genome achieve their functions by binding the C-termini of other proteins in sequence-specific manners. We have used high-throughput technologies to investigate the biological roles of PDZ domains in colorectal cancer, evaluate the prevalence of PDZ domains that interact directly with other PDZ domains, and understand the biophysical basis for C-termini recognition. The role of PDZ domains in colorectal cancer was found through an unbiased, family-wide, protein microarray-based screen of all PDZ domains against the C-terminus of β-catenin, a ubiquitously expressed, multifunctional protein that plays important roles in cell adhesion and signal transduction. β-catenin-PDZ domain interactions were found to be important in the proper maintenance of cell-cell junctions, the integrity of which is related to cancer. A similar family-wide investigation of PDZ-PDZ interactions revealed many new interactions of biological interest. Concurrently, through the use of high-throughput fluorescence polarization assays, we successfully constructed a model that is able to accurately predict the binding energies of PDZ domains to any C-terminal amino acid sequence. We demonstrate how this model can be used to predict biologically relevant PDZ-protein interactions and to design selective, high affinity peptide inhibitors that block PDZ interactions in a cellular context. Following the success of this model, we developed a high-throughput platform that can deliver a variety of biomolecules, including peptides, directly into cells. Together, these studies highlight the importance of PDZ domains in biology while providing the foundation for the future high-throughput examination of their critical biological roles. Ultimately, this work provides the tools to probe, oneby-one, the function of each PDZ domain in the genome and learn about disease-relevant PDZ domain interactions.