Bicyclic peptides are small, constrained peptides that can bind with high affinity and selectivity to protein targets. Their small size provides a number of advantages over larger protein-based ligands, including access to chemical synthesis, better tissue penetration, and a wider choice of application routes. Bicyclic peptide ligands can be identified using phage display technology with moderate effort and cost. Here we provide step-by-step protocols for the isolation of bicyclic peptide ligands using phage display. These protocols have been successfully used in our laboratory for the generation of high-affinity binders to a variety of protein targets. We describe library generation, affinity selection and ligand characterization, and provide troubleshooting advice concerning frequent problems. (C) 2013 Elsevier Inc. All rights reserved.