Covipyte™ EN450 is a peptide substrate containing 9 amino acid sequence (RELNGGAPI) that can be cleaved by coronavirus PLpro. The dark-FRET peptide contains Edans (donor) and Dabcyl (quencher) on the N-and C-terminals respectively where the fluorescence of Edans is effectively quenched by Dabcyl when the peptide is intact. When the peptide is hydrolysed by coronavirus proteases, the Edans fragment generates significantly enhanced fluorescence since its fluorescence is no longer quenched by Dabcyl. The activity of coronavirus proteases can be effectively monitored by the fluorescence intensity of Edans. Covipyte™ EN450 is a convenient tool for screening and studying kinetics of PLpro inhibitors.