The COVID-19 pandemic has caused significant losses to the global community. Although effective vaccination and antiviral therapeutics provide primary defense, SARS-CoV-2 remains a public health threat, given the emerging resistant variants. The SARS-CoV-2 papain-like protease (PL) is essential for viral replication and is a promising drug target. We recently designed a series of biarylphenyl PL inhibitors with a representative lead showing potent antiviral efficacy in a SARS-CoV-2 infection mouse model. In this study, we designed a fluorescein-labeled biarylphenyl probe and used it to optimize a fluorescence polarization (FP) assay. The FP assay is suitable for high-throughput screening with a ' factor of 0.69. In addition, we found a positive correlation between the FP binding affinity and the enzymatic inhibitory potency of PL inhibitors, suggesting that the FP assay is valid in characterizing the binding affinity of PL inhibitors.