Perfluorinated surfactants are demonstrated to dramatically enhance desorption/ionization on fluorinated silicon (DIOS) mass spectrometry. Perfluorooctanesulfonic acid improved the signal-to-noise ratio of tryptic digests and gave a 3-fold increase in the number of peptides identified. Similar results were also obtained using perfluoroundecanoic acid; yet among the seven different surfactants tested, controls such as nonfluorinated sodium dodecyl sulfate or fluorinated molecules with minimal surfactant activity did not enhance the signal. The same surfactants also enhanced the DIOS-MS signal of amino acids, carbohydrates, and other small organic compounds. The signal enhancement may be facilitated by the high surface activity of the perfluorinated surfactants on the fluorinated silicon surfaces allowing for a higher concentration of analyte to be absorbed.