AbstractPerfluorocarbons, saturated carbon chains in which all the hydrogen atoms are replaced with fluorine, form a separate phase from both organic and aqueous solutions. Though perfluorinated compounds are not found in living systems, they can be used to modify biomolecules to confer orthogonal behavior within natural systems, such as improved stability, engineered assembly, and cell‐permeability. Perfluorinated groups also provide handles for purification, mass spectrometry, and 19F NMR studies in complex environments. Herein, we describe how the unique properties of perfluorocarbons have been employed to understand and manipulate biological systems.