Introduction: Elimination of viral hepatitis as a major public health threat by 2030 was announced by the Global Health Sector Strategy (GHSS) on viral hepatitis in 2016. Hepatitis C is one of the major causes of liver cirrhosis and liver cancer. Complications as a results of hepatitis C infection can be prevented as hepatitis C infection is now con-sidered as a curable disease with the availability of Direct Acting Agents (DAAs). However, the main barrier towards treating and curing all HCV infected patients is a high cost of DAAs. The treatment regime of hepatitis C infection in Malaysia is sofosbuvir and daclastavir, an NS5A inhibitor. Daclastavir was reported as inherently resistant to HCV GT 3. Thus, this study aimed to develop an assay to detect the resistance associated substitution (RAS) towards the NS5A inhibitor among HCV GT 3 infected patients. Methods: Samples for the study were obtained from various hospitals in Malaysia. The samples were collected from DAAs naïve HCV GT 3 infected patients. From the literature review, a specific assay was chosen with different sets of primers were selected for the study. The DNA sequences of NS5A region of HCV genome were submitted to the geno2pheno [HCV] resistance database by Max Planck Institut (MPI) Informatics to yield RAS. Results: Suitable primers were identified based on generated amplicons produced for the samples which NS5A region were successfully sequenced. Results were obtained based on the 213 codon generated from the population based Sanger sequencing. RAS/ RASs towards Daclastavir were produced and the susceptibility result towards the drug was generated. Conclusion: The assay was successfully optimised and able to generate drug resistance results towards Daclastavir which might have impacts on the duration of treatment and/or inclusion of ribavirin in managing HCV infected patients in Malaysia.