Purpose: In vivo screening of phage-displayed random peptide libraries (RPLs) has been used to identify peptide ligands to targets found on endothelial cells of blood vessels supplying specific tissues such as brain, kidney, and tumor tissue. Peptides that bind specifically to blood vessels supplying tumor tissue have been conjugated to cytotoxic agents and used to successfully eradicate tumors in a mouse model. With the ultimate goal of developing similar methods for treating human cancer, we describe an in vivo RPL screening process that, unlike previous in vivo experiments, does not harm the animal being screened. Methods: RPLs were administered to FVB, BalbC, and tumor-bearing MRL/MpJ-fasLPR mice in a variety of dosing formats. Tumor nodules were excised 10 min following infusion and phage were amplified from the specimens. Phage were reinjected into the same animal within 48 h. This process was repeated twice for a total of three in vivo screens of mouse tumor tissue within the same animal. Mice were observed for systemic side effects, histopathologic damage, and presence of phage in organs. Peptide sequences were determined from several third-pan phage clones. Results: Overall there was minimal toxicity from administration of single or repeat doses of RPLs. Amino acid consensus sequences were identified and some of the sequences were similar to those of peptide ligands that bind matrix metalloproteinases. Conclusions: Serial administration of an RPL is well tolerated and serial panning in individual mice leading to consensus sequence motifs is possible. Based on these preclinical data the Food and Drug Administration has approved the implementation of human clinical trials with this technique.