Hepatitis C infection has become a leading cause of chronic liver disease and a globally growing public health concern these days. It has infected about 150 million individuals worldwide including ten millions in Pakistan. The standard protocol to treat the Hepatitis C patient is based on the confirmation of the Hepatitis C virus (HCV) RNA in the patient’s blood, determination of viral load and its specific genotype. This information helps to decide the nature and duration of therapy. Before treating the Hepatitis C patients, an in-house HCV RNA qualitative polymerase chain reaction (PCR) test for the detection of HCV on molecular level was established. To investigate the specific genotype of HCV in the patient, a multiplex HCV genotyping assay was also established. HCV RNA was detected in 4981 (75.81%) out of 6570 cases which were reactive for Anti-HCV antibody test by ELISA method. 4528(90.90%) of the HCV RNA positive patients had single genotype, 201(4.04%) had more than one genotypes whereas in 252(5.06%) HCV remained non-classifiable. Although genotype 3 was the most frequent type in the present study (68.10%). However, a downward trend was observed in the prevalence of genotype 3 in last five years (2007-2012). On the contrary, the prevalence of genotype 1 and 4 was observed to be rising during that period. Transmission of HCV was strongly associated with the use of un- sterilized razors in barber shops as well as in those who underwent procedures in general or dental surgery. The low titer of HCV RNA at the start of therapy, more than two log drop of viral load at week-4 and undetectable HCV RNA in the middle of therapy were associated with the response of HCV infected patients treated with Interferon alpha 2b plus Ribavirin (IFNα- 2b+RBV). So, the findings of present study suggest that the viral load measurement at different time points during the therapy could be used in clinical practice to decide the treatment continuation or termination at early stages. The response to treatment (IFNα-2b+RBV) was also observed to be inversely related to the age of the patient. Younger age was associated with a better response and old age with treatment failure. The ratio of ETR was marginally higher in males (76.14%) than females (72.77%). Sustained response was observed to be high in genotype 3. The treatment response was also highly associated with amino acid sequence variation in both the selected regions of E2 protein from HCV subtype 3a (E2 506-564 and E2 622-714 ).