The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, we tested the recovery of radiolabeled 125I endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. 125I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-1 GLP-1, insulin, leptin, nesfatin-1, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of 125I-labeled peptides was determined by gamma counting. Important differences in 125I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing 125I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced 125I-ghrelin from borosilicate glass, whereas GLP-1 and Fmoc-arginine did not. Choosing the appropriate experimental container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions.