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The fluorescence resonance energy transfer (FRET) assay is widely used for high\u2010throughput screening (HTS) of Mpro inhibitors, but there has been a significant increase in false positives stemming from flawed assay design in previous studies. Here, we provide an overview of the FRET assay, discuss the key points of this method design, and highlight the corresponding solutions. We hope that this issue should receive increased attention from researchers.<\/jats:p>","DOI":"10.1002\/jmv.70801","type":"journal-article","created":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T09:37:56Z","timestamp":1768383476000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Fluorescence Resonance Energy Transfer Assay at the Crossroad: Urgent Reexamination of Assay Design for Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors"],"prefix":"10.1002","volume":"98","author":[{"given":"Jiankai","family":"Ye","sequence":"first","affiliation":[{"name":"Laboratory for Drug Screening and Evaluation, Wannan Medical College, Wuhu  China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Xu","sequence":"additional","affiliation":[{"name":"Laboratory for Drug Screening and Evaluation, Wannan Medical College, Wuhu  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