This chapter describes the application of ionic liquids (ILs) in analytical chemistry including their applications in gas chromatography, high-performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectra, and capillary electrophoresis, as well as enrichment of n-3 polyunsaturated fatty acids/esters. Specifically, the application of ILs in lipid analysis/separation are highlighted.