Simeprevir (TMC435) is a once-daily, single-pill, oral hepatitis C virus (HCV) NS3 protease inhibitor approved for the treatment of chronic HCV infection. Phenotypic characterization of baseline isolates and isolates from HCV genotype 1-infected patients failing with a simeprevir-based regimen was performed using chimeric replicons carrying patient-derived NS3 protease sequences. Cutoff values differentiating between full susceptibility to simeprevir (≤ 2.0-fold reduction in simeprevir activity) and low-level versus high-level resistance (≥ 50-fold reduction in simeprevir activity) were determined. The median simeprevir fold change in the 50% effective concentration (FC) of pretreatment genotype 1a isolates, with and without Q80K, and genotype 1b isolates was 11, 0.9, and 0.4, respectively. Naturally occurring NS3 polymorphisms that reduced simeprevir activity, other than Q80K, were uncommon in the simeprevir studies and generally conferred low-level resistance in vitro. Although the proportion of patients with failure differed by HCV geno/subtype and/or presence of baseline Q80K, the level of simeprevir resistance observed at failure was similarly high irrespective of type of failure, HCV genotype 1 subtype, and presence or absence of baseline Q80K. At the end of the study, simeprevir activity against isolates that lost the emerging amino acid substitution returned to pretreatment values. Activity of simeprevir against clinical isolates and site-directed mutant replicons harboring the corresponding single or double amino acid substitutions correlated well, showing that simeprevir resistance can be attributed to these substitutions. In conclusion, pretreatment NS3 isolates were generally fully susceptible (FC, ≤ 2.0) or conferred low-level resistance to simeprevir in vitro (FC, >2.0 and <50). Treatment failure with a simeprevir-based regimen was associated with emergence of high-level-resistance variants (FC, ≥ 50).