Proteolytic enzymes are involved in many important physiological processes. Because of the critical roles played by these enzymes, aberrations in regulation of their activities can lead to pathological conditions. For this reason, finding inhibitors selective for a proteolytic enzyme that is contributing to a medical problem can be an effective therapeutic strategy. The introduction of fluorine in the backbone of proteolytic enzyme substrates can lead to active and selective inhibitors belonging to the peptidyl fluorinated ketone family. Fluorine not only can influence the mechanism of substrate/enzyme recognition events but also can modify the in vivo profile of the substrate. Although prediction of the total effects of fluorine on the pharmacokinetic parameters can be difficult, the pharmaceutical interest in the synthesis and biological evaluation of peptidyl fluorinated ketones highlights the potential of this family of molecules as therapeutically useful inhibitors.