The post-transitional modification of peptidyl arginine to citrulline by PAD2 can affect the inherent biophysical properties of the citrullinated protein. Furthermore, dysregulation of PAD2 activity has been implicated in a number of human diseases. Inhibition of these enzymes by small molecules can serve as essential probes in establishing a link to pathogenesis. Herein, we describe a profluorescent substrate analog that reports on the activity and the inhibition of PAD2 in a robust assay. Most noteworthy, we expect future drug discovery efforts based on PAD2 inhibition can be pursued via this assay.