Chromatographic measurements were made on 17 physicochemically diversified high-performance liquid chromatography (HPLC) columns which were further analyzed in terms of their similarities and dissimilarities for 25 carefully designed, structurally diverse peptides showing distinctly distinguished groups. The goal of the study was to investigate the molecular mechanism of retention and to find an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for reverse phase HPLC (RP-HPLC). We utilized the structural descriptors of peptides obtained from molecular modeling to describe their chromatographic retention behavior under given HPLC conditions. Quantitative structure-retention relationships (QSRR) with the following descriptors were employed: logarithm of the sum of amino acid retention contributions in a given peptide, logSumAA, logarithm of the Van der Waals volume of the peptide, logVDWvol, and logarithm of its calculated n-octanol/water coefficient, clogP. The best QSRR equations were obtained in the case of monolithic and regular octadecylsilica columns. On the other hand, the combination of QSRR and principal component analysis (PCA) can be considered as the efficient tool allowing column classification and searching for orthogonal HPLC conditions required to separate peptides.