Perfluoroalkyl-containing compounds are highly fluorous, meaning that they have a remarkable affinity for one another and effectively exclude non-fluorous species. Utilizing this unique property, we have developed a fluorous derivatization with a liquid chromatographic analysis method for highly selective analysis of target analytes. Although most previous methods focused on extremely sensitive detection-oriented derivatization, the fluorous derivatization method involves highly specific separation for analytes. This method includes perfluoroalkylation of analytes with a fluorous reagent, and separation of the derivatives using a perfluoroalkyl-modified stationary phase LC column. The derivatives can be selectively retained on the fluorous-phase LC column, whereas the non-fluorous derivatives are poorly retained under the same separation conditions. The combination of this method with LC-tandem mass spectrometry (MS/MS) is very useful for complex biological sample analysis, because matrix-induced suppression effects, which are a common problem in LC-MS/MS analysis arising from components of a biological endogenous matrix, have not been observed. We have successfully applied this method to precise and accurate LC-MS/MS analysis of some biogenic compounds, such as sialic acids and biogenic amines, in complex biological samples.