The main protease (Mpro) is a promising drug target for inhibiting the coronavirus due to its conserved properties and lack of homologous genes in humans. However, previous studies on Mpro’s kinetic parameters have been confusing, hindering the selection of accurate inhibitors. Therefore, obtaining a clear view of Mpro’s kinetic parameters is necessary. In our study, we investigated the kinetic behaviors of Mprofrom SARS-CoV-2 and SARS-CoV using both FRET-based cleavage assay and the LC-MS method, respectively. Our findings indicate that the FRET-based cleavage assay could be used for preliminary screening of Mproinhibitors, while the LC-MS method should be applied to select the effective inhibitors with higher reliability. Furthermore, we constructed the active site mutants (H41A and C145A) and measured the kinetic parameters to gain a deeper understanding of the atomic-level enzyme efficiency reduction compared to the wild type. Overall, our study provides valuable insights for inhibitor screening and design by offering a comprehensive understanding of Mpro’s kinetic behaviors.