A comprehensive two‐dimensional liquid chromatography system was developed to precisely describe the molecular heterogeneity of fatty alcohol ethoxylates. The end‐group functionality was analyzed by gradient HPLC while ethylene oxide oligomer distributions were characterized by liquid adsorption chromatography. A baseline separation of all functionality fractions irrespective of the ethylene oxide oligomer chain length was achieved on nonpolar X‐Terra® C18 with a methanol–water gradient, whereas an isocratic flow of isopropanol–water on a polar Chromolith® Si column gave a separation according to the oligomer chain length without interference of the end‐group distribution. The combination of these two methods to conduct online two‐dimensional liquid chromatography experiments resulted in a comprehensive two‐dimensional picture on the molecular heterogeneity of the sample.