Polysorbates can be separated according to their functionality and architecture by liquid chromatography on a hydrophilic interaction chromatography (HILIC) column in acetone-water mobile phases containing 90-97% acetone. The different polymer homologous series are separated according to the number of terminal hydroxy groups and elute as narrow peaks. The hydrophilic part (ethoxylates of sorbitan, isosorbide, and poly(ethyleneglycol)) and amphiphilic funtionalites (ethoxylated mono-, di-, tri-, and tetraesters) were separated by HILIC mode of high-performance liquid chromatography (HPLC). All these separated functionaities are identified and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). This combination of HPLC and MALDI-TOF MS has been proven to be an excellent tool for the characterization of heterogenous complex samples.