A systematic study has been made of peptide retention in reversed phase and hydrophobic interaction chromatography. Measurements have been made for a range of peptides under varied surface hydrophobicity, peptide structure, peptide charge, cosolvent concentration, temperature, pH, and concentration of added salt. The conditions and peptides have been organized so that, for at least three peptides, only a single variable is changed. These stepwise variations should provide benchmark data to validate molecular models and computer simulations that predict adsorption and retention in hydrophobic media from biochemical structure and a minimum of adjustable parameters.