In vivo phage display is a technology used to reveal organ-specific vascular ligand-receptor systems in animal models and, recently, in patients, and to validate them as potential therapy targets. Here, we devised an efficient approach to simultaneously screen phage display libraries for peptides homing to any number of tissues without the need for an individual subject for each target tissue. We tested this approach in mice by selecting homing peptides for six different organs in a single screen and prioritizing them by using software compiled for statistical validation of peptide biodistribution specificity. We identified a number of motif-containing biological candidates for ligands binding to organ-selective receptors based on similarity of the selected peptide motifs to mouse proteins. To demonstrate that this methodology can lead to targetable ligand-receptor systems, we validated one of the pancreas-homing peptides as a mimic peptide of natural prolactin receptor ligands. This new comprehensive strategy for screening phage libraries in vivo provides an advantage over the conventional approach because multiple organs internally control for organ selectivity of each other in the successive rounds of selection. It may prove particularly relevant for patient studies, allowing efficient high-throughput selection of targeting ligands for multiple organs in a single screen.--Kolonin, M. G., Sun, J., Do, K.-A., Vidal, C. I., Ji, Y., Baggerly, K. A., Pasqualini, R., Arap, W. Synchronous selection of homing peptides for multiple tissues by in vivo phage display.