AbstractThe post‐translational modifications of histone proteins are highly diverse and dynamic processes. It is becoming increasingly evident that modifying histone proteins can have a direct influence on both cellular homeostasis and disease states. Protein arginine deiminase 4 (PAD4) is an enzyme that converts peptidyl‐arginine to citrulline. The overexpression of PAD4 has been found in numerous types of human cancer and autoimmune diseases. We report a new, facile, fluorescence‐based assay for the detection of PAD4 activity that exploits the substrate specificity of trypsin to monitor the citrullination reaction carried out by PAD4 based on the fact that, upon citrullination, the positively charged arginine side chain is converted to the neutral citrulline. We show that the assay can be performed rapidly with readily available reagents and that it responds accordingly to a known PAD4 inhibitor.