Surfactants suppress the electrospray ionization (ESI) of various compounds. Here we demonstrate that fluorinated surfactants such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOSA) could be employed for ESI-MS without a significant decrease in sensitivity. Both PFOA and PFOSA could be applied to the analysis of ionic and nonionic compounds by micellar electrokinetic chromatography (MEKC)/ESI-MS, although the migration window was limited. Furthermore, in HPLC/ESI-MS, PFOA could function as a paired ion for enzymatically digested peptides as well as sulfonamides and significant effects of PFOA on the signal peak shape, retention times, and sensitivity of the analytes were observed compared to those of trifluoroacetic acid or sodium dodecyl sulfate (SDS). In addition, PFOA was applied to protein analysis by ESI-MS, and superior sensitivity was noted, compared to other surfactants such as octylglucoside, dodecylglucoside, and SDS. Although 21% of the original signal was observed in the presence of 1% PFOA, this surfactant could be easily removed by evaporation, which resulted in the recovery of 96% of the original signal. Because these fluorinated surfactants could also be used for solubilization, extraction, and disaggregation of proteins, they should greatly expand the applicability of the ESI-MS to the biological problems of proteins.