Two-dimensional HPLC was applied for the first time to the analysis of complex surfactant mixtures. In the first dimension, ion chromatographic-type separations were performed on a diol column eluted by an acetonitrile-water (0.1% trifluoroacetic acid) gradient. Using this new technique, cationic and amphoteric surfactants were not retained at all, nonionic surfactants exhibited a weak and essentially unspecific retention, and anionic surfactants were retained mainly according to their functional group. Rather than detecting the analytes immediately after this first separation, successive fractions were automatically and quantitatively transferred to parallel C2 (dimethyl) and C4 (butyl) reversed-phase columns using an innovative setup. The second dimension of the separation then took place, by which the analytes were separated according to their hydrophobicity. Surfactants from all four classes, cationic, amphoteric, nonionic, and anionic, were separated simultaneously in single 54-min two-dimensional HPLC runs. The suitability of the method for quantitative measurements was demonstrated.