Backround: The effect of methadone (MET) during therapy with novel direct-acting antiviral agents is still not fully understood. Currently, no data are available about the influence of MET on daclatasvir (DCV) plasma levels in patients affected by chronic hepatitis C (CHC). The aim of this study was to assess the DCV plasma concentrations in patients treated with sofosbuvir (SOF) plus DCV, with or without ribavirin (RBV) and with or without MET. Methods: In this analysis, 47 patients were included, treated consecutively with SOF + DCV ± RBV for 24 weeks, from May to October 2015; 22 (46.8%) received MET substitutive therapy. Results and Conclusion: We found a significant difference in DCV levels at 2 weeks and 1 month: 150 ng/mL in patients without MET and 313 ng/mL with MET at 2 weeks (p < 0.001), 149 and 279 ng/mL at 1 month (p = 0.006). DCV levels were lower in cirrhotic patients (p < 0.001); among cirrhotic patients we also evidenced higher DCV concentrations in patients receiving MET at 2 weeks, 1 and 2 months (p < 0.001, p = 0.005, and p = 0.031, respectively). In multivariate analysis, the only predictive factor associated with DCV plasma levels was the presence of MET. The reason for this increased DCV exposure is unclear; on the clinical side, we have not observed significant adverse events related to the reduction or increase of MET plasma levels. The administration of MET in patients with advanced fibrosis or cirrhosis leads to an early increase of DCV plasma level without significant clinical effects or toxicity.