In patients with hepatitis C virus (HCV), treatment failure of Daclatasvir plus Asunaprevir combination therapy (DCV+ASV) seems to become intractable due to the induction of resistance-associated substitutions (RASs). This study aimed to investigate the outcomes of retreatment with direct-acting antivirals (DAAs) in patients with DCV+ASV therapy failure as well as changes in drug resistance mutations. We retrospectively analyzed 44 patients re-treated with DAAs following DCV+ASV failure between December 2015 and April 2018. All patients were analyzed for amino acid substitutions (AASs) and additional treatment regimens were selected based on the results and current treatment guidelines. The SVR rate with second-line treatment was 81.8% (36/44), and relapse occurred in 5 of 16 patients who received Sofosbuvir/Ledipasvir (SOF/LDV) and 3 of 7 patients who received DCV/ASV/Beclabuvir (DCV/ASV/BCV). Third- and fourth-line treatments were also tried in relapsed cases, and the overall SVR rates were 90.9% (40/44) and 93.2% (41/44), respectively. A high rate of viral clearance was eventually observed. Prior to second-line treatment, the prevalence of mutations in the NS5A and NS3/4A regions was 100% (44/44) and 86.4% (38/44), respectively. There was no significant increase in the number of AASs in patients in whom second-line treatment failed. AASs were frequently observed in patients with DCV+ASV failure, but most patients achieved an SVR following re-treatment with DAAs. Although the spread of drug-resistant viruses due to unsuccessful DAA treatment was a matter of concern, most cases of DCV+ASV failure were overcome with additional treatment. This article is protected by copyright. All rights reserved.