Rupintrivir (formerly AG7088) is an irreversible inhibitor of the human rhinovirus (HRV) 3C protease that has been demonstrated to have in vitro activity against all HRVs tested, consistent with its interaction with a strictly conserved subset of amino acids in the 3C protease. The potential for resistance was studied following in vitro serial passage of HRV serotypes 14, 2, 39, and Hanks in the presence of increasing rupintrivir concentrations. HRV variants with reduced susceptibilities to rupintrivir (sevenfold for HRV 14) or with no significant reductions in susceptibility but genotypic changes (HRV 2, 39, and Hanks) were initially isolated following 14 to 40 cumulative days in culture (three to six passages). Sequence analysis of the 3C protease identified one to three substitutions in diverse patterns but with common features (T129T/A, T131T/A, and T143P/S in HRV 14; N165T in HRV 2; N130N/K and L136L/F in HRV 39; T130A in HRV Hanks). Notably, three of the four HRV variants contained a substitution at residue 130 (residue 129 in HRV 14). Continued selection in the presence of escalating concentrations of rupintrivir (40 to 72 days) resulted in the accumulation of additional mutations (A121A/V and Y139Y/H in HRV 14, E3E/G and A103A/V in HRV 2, S105T in HRV 39), with only minimal further reductions in susceptibility (up to fivefold). The ability of specific substitutions to confer resistance was examined by susceptibility testing of HRV 14 variants constructed to contain 3C protease mutations. In summary, the slow accumulation of multiple amino acid substitutions with only minimal to moderate reductions in susceptibility highlight the advantages of 3C protease as an antiviral target.