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Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.