Peptide aptamers are combinatorial proteins that specifically bind intracellular proteins and modulate their function. They are powerful tools to study protein function within complex regulatory networks and to guide small-molecule drug discovery. Here we describe methodological improvements that enhance the yeast two-hybrid selection and characterization of large collections of peptide aptamers. We provide a detailed protocol to perform high-efficiency transformation of peptide aptamer libraries, in-depth validation experiments of the bait proteins, high-efficiency mating to screen large numbers of peptide aptamers and streamlined confirmation of the positive clones. We also describe yeast two-hybrid mating assays, which can be used to determine the specificity of the selected aptamers, map their binding sites on target proteins and provide structural insights on their target-binding surface. Overall, 12 weeks are required to perform the protocols. The improvements on the yeast two-hybrid method can be also usefully applied to the screening of cDNA libraries to identify protein interactions.