Hepatitis C is a major health problem affecting more than 200 million individuals in World including Pakistan. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed to eliminate virus completely from the patient’s body. The mechanism how Hepatitis C Virus (HCV) induces interferon resistance is still indefinable. HCV genotype 1a shows greater hindrance to treatment than genotype 3a. One of the mechanisms by which virus evades the antiviral effect of interferon alpha involves that HCV envelope protein 2 (E2) interacts with Protein Kinase (PKR) which is the interferon-inducible protein kinase and which in turn blocks the activity of its target molecule called eukaryotic translation initiation factor 2 alpha (eIF2α). Sequence analysis of Envelope protein reveals it contains a domain homologous to phosphorylation sites of PKR and the translation initiation factor eIF2alpha. This domain is known as protein kinase (PKR) eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation homology domain (PePHD). This domain in HCV genotype 1 strains is reportedly homologous to PKR and its target eIF2α. Thus envelope protein competes for phosphorylation with PKR. By binding to PKR, PePHD inhibits its activity and therefore cause virus to evade antiviral activity of interferon (IFN). In the present study the possible role of phosphorylation in envelope 2 protein for interferon resistance was first investigated in silico and then confirmed invivo. Genes coding for envelope 2 protein were isolated from local HCV isolates and their tertiary structure was predicted. Insilico phosphorylation of tertiary structures revealed that two residues S75 and S277 of envelope 2 gene are surface exposed at cytoplasmic domain and may compete with the phosphorylation of PKR protein. Interferon induced antiviral protein PKR has a role in the HCV treatment as dsRNA activated PKR has the capacity to phosphorylate the serine and threonine of E2 protein and dimerization of viral RNA. E2 gene of HCV genotype 1 has an active role in IFN resistance. E2 protein inhibits and terminates the kinase activity of PKR by blocking it in protein synthesis and cell growth. This brings forward a possible relation of E2 and PKR through a mechanism via which HCV evades the antiviral effect of IFN. A hybrid in-silico and wet laboratory approach of motif prediction, evolutionary and structural analysis has pointed out serine 75 and 277 of the HCV E2 gene as a promising candidate for the serine phosphorylation. It is proposed that Serine phosphorylation of HCV E2 gene has a significant role in interferon resistance. In present study we mutated the two Serineresidues at positions S75 and S277 and their efficacy was checked in respect to interferon resistance. The results of this study suggest that serine residues as predicted have a significant role in interferon resistance, especially S75. It is also suggested that some other factors may be involved along with viral envelope gene 2.