BACKGROUND & AIMS PCR-Invader is a highly sensitive assay for detecting nonstructural protein 5A (NS5A) resistance-associated variants (RAVs) of hepatitis C virus (HCV). Here, we validated the accuracy of the semiquantitative PCR-Invader (SQ-PI) assay compared to direct sequencing (DS) for identifying NS5A RAVs, and we evaluated the treatment efficacy of daclatasvir plus asunaprevir (DCV+ASV) for patients judged to be nonpositive for NS5A RAVs by SQ-PI. METHODS Detection rates of NS5A RAVs by SQ-PI and DS were compared for 204 patients with HCV genotype 1b. Patients with nonpositive results for NS5A RAVs by SQ-PI were treated by DCV+ASV, and the efficacy of this treatment was examined. RESULTS All samples judged as negative for NS5A RAVs by SQ-PI were similarly judged by DS. However, 29.7% of samples judged as negative for Y93H by DS were judged as weakly positive or positive by SQ-PI. Among 108 patients who were judged as negative by SQ-PI and treated by DCV+ASV, rates of sustained virologic response at 24 weeks (SVR24) were 96.3% in intention-to-treat analysis and 99.0% in patients who completed treatment. Among patients who were weakly positive for Y93H on SQ-PI, the SVR24 rate was 95.0% (19/20). This rate was 100% (78/78) in patients who were negative for Y93H on SQ-PI and completed treatment. CONCLUSION Treatment efficacy of DCV+ASV was extremely high among patients who were nonpositive for NS5A RAVs on SQ-PI, especially for patients with negative results.