Protein-protein interactions (PPIs) play important roles in cellular processes and the stabilization of complexes is performed by amino acids at the interaction interface with steric and energetic complementarity. Although methods such as X-Ray Crystallography and Nuclear Magnetic Resonance are used to identify interfacial residues in PPI, they are extremely laborious, expensive, and require specialized equipment and data analysis experts. This chapter describes a rapid and low-cost protocol for large-scale screening of potential interfacial peptides in PPI based on the adaptation of the enzyme-linked immunosorbent assay (ELISA) with a previous step of protein depletion using microplates containing different peptide sequences. The peptides to be tested can be chemically synthesized containing continuous amino acid sequences or by mimicking discontinuous regions favored by the spatial arrangement in the protein conformation. The depletion ELISA works in low concentrations of proteins with high sensitivity and specificity and potential to screen several peptides in 30 h.