PLpro of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2 PLpro, also referred to as SARS-CoV-2 Papain-like protease, Nsp3) plays an essential role in processing the polyproteins 1ab for viral replication. Inhibition of this enzyme activity would block viral replication, making it a promising target for anti-coronaviral therapeutic agents. The assay is fast and convenient and requires just two steps. In the first step, the PLpro enzyme is preincubated with the compound for 30 minutes. The reaction is initiated by adding substrate solution at the second step. Fluorescent intensity is measured with a fluorescent plate reader at the excitation wavelengths of 340-360 nm and emission wavelengths of 460-480 nm.