High-throughput sequencing was previously applied to phage-selected peptides in order to gain insight into the abundance and diversity of isolated peptides. Herein we developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides for the purpose of identifying target-binding peptide motifs. We applied the procedure to analyze bicyclic peptides isolated against five different protein targets: sortase A, urokinase-type plasminogen activator, coagulation factor XII, plasma kallikrein and streptavidin. We optimized sequence data filters to reduce biases originating from the sequencing method and developed sequence correction algorithms to prevent identification of false consensus motifs. With our strategy, we were able to identify rare target-binding peptide motifs, as well as to define more precisely consensus sequences and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and it facilitates identification of epitopes. We furthermore show that binding motifs can be identified after a single round of phage selection. Such a selection regimen reduces propagation-related bias and may facilitate application of phage display in non-specialized laboratories, as procedures such as bacterial infection, phage propagation and purification are not required.