The oral small-molecule drug nirmatrelvir (NTV)1 is now widely distributed as combinational COVID-19 therapeutics (along with ritonavir) by the brand name Paxlovid, targeting one of the major enzymes responsible for SARS-CoV-2 viral replication, non-structural protein 5 (nsp5), also known as the main protease (Mpro). Paxlovid has shown positive therapeutic effects on patients suffering from mild-to-moderate COVID-19 with risk of progression to severe disease conditions. However, with increased administration (more than 1.7 million doses) of paxlovid across the globe, there is an alarming possibility of future SARS-CoV-2 lineages evading the inhibitor by acquiring resistant mutations in Mpro. Early prediction and evaluation of these resistance mutations is, therefore, critical in the development of newer and potent anti-viral drugs with activity against the resistant strains. In the present work, we have utilized in silico mutational scanning and substrate-inhibitor covalent docking against Mpro to identify potential resistance mutations2. Mutational scanning was directed at residues, forming and neighbouring the active site, to generate 3 sets of mutant libraries, while covalent docking was performed using the substrate (SAVLQSGF) and inhibitor (NTV) successively to screen out models producing poor substrate and good inhibitor binding orientation. Subsequent in vitro experiments3 revealed mutations N142L, E166M, Q189E, Q189I, and Q192T reduced the potency of NTV and a previously identified potent macrocyclic peptide inhibitor of Mpro (Peptide I)4. The E166M mutant especially reduced the half-maximal inhibitory concentration (IC50) of NTV by 24-fold and 118-fold for Peptide-1. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages.