Peptide aptamers are small recombinant proteins typically inserted into a supportive protein scaffold. These short peptide domains can bind to their target proteins with high specificity and affinity, often resulting in an altered target protein. We describe high-throughput protocols that facilitate the selection and characterization of peptide aptamers from yeast dihybrid libraries. These protocols include the preparation and evaluation of the bait fusion and the peptide aptamer screen. They also include confirmation of interaction specificity as well as isolation and sequencing of peptide inserts. Once the amino acid sequence is determined, we describe a protocol for aligning and comparing short peptide sequences and assessing the statistical significance of the alignments.