Hepatitis C virus (HCV) response to direct-acting antivirals (DAAs) may be influenced by the presence of resistance-associated substitutions (RASs). This study aimed to assess if NS5A baseline RAS-guided treatment enhances the rate of sustained viral response (SVR) in naïve HCV-infected patients in clinical practice. All HCV-infected patients who initiated treatment with interferon (IFN)-free DAA-based regimens between March 2016 and May 2017 in 17 Spanish hospitals and who had evaluable SVR 12 weeks (SVR12) after the end of therapy were included. Patients had to be DAA naïve, with the exception of sofosbuvir with/without IFN. In one hospital, participants received therapy guided by the presence of NS5A-RASs (RGT population). Patients enrolled in the remaining hospitals, without baseline RASs testing, constituted the control population. A total of 120 and 512 patients were included in the RGT and control populations, respectively. Nine (7.5%) individuals in the RGT population showed baseline NS5A-RASs. All of them achieved SVR12. The SVR12 rate in the RGT population was 97.2% (three relapses) whereas it was 98.8% (six relapses) in the control population (p = 0.382). Our findings suggest that testing for baseline NS5A-RASs in naïve HCV-infected patients does not enhance the rate of SVR to DAA-based IFN-free therapy in clinical practice.