The retention and separation of ten iridoid glucosides typical for the plants of genusGalium(Rubiaceae) were examined by RP-HPLC on octadecyl (ODS) column with water-methanol mobile phases. The compounds have the same basic carbon skeleton with a glucose moiety at C-1 but differ by the type, the number, and the position of the additional functional groups. In general, the retention increased with the increasing total hydrophobicity of the molecule, but was substantially affected by the different combinations of free and esterified hydroxyl and /or carboxyl groups. The water-based mobile phase, the high polarity and the bulky structure of the iridoid glucosides examined suggested that the solute-mobile phase interactions dominated in the retention mechanism. Water-methanol (85:15, v/v) was found to be a suitable mobile phase for the fractionation of the iridoid glucosides into acidic and non - acidic components on a single column.