Hepatitis C virus (HCV), a single-stranded RNA virus belonging to the Flaviviridae family, has been identified as a major pathogen of post transfusion and community- transmitted non-A, non-B hepatitis. One limitation in HCV research is the lack of a highly sensitive and specific assay to measure viral loads in plasma or serum. HCV circulates in the blood at a low copy number and its genome is extremely heterogeneous. Many investigators used different methods to quantify the viral load in HCV-infected patients. Recently, a real-time RT-PCR analysis has been employed successfully for both basic research and clinical applications. In the present work, 72 patients were studied on referral to the molecular biology research unit of Assuit university, Egypt for HCV quantification by real-time PCR. Initially, all patients were screened by enzyme linked immunosorbent assay (ELISA) for the presence of anti-HCV antibodies. Six cases (8.3%) were anti-HCV positive and 66 cases (91.7%) were non-reactive for HCV- antibodies in their sera. Real-time RT polymerase chain reaction assay for quantification of hepatitis C virus (HCV) RNA in the sera of all patients was carried out using a pair of primers and Taq-man probe that are specific for recognsion of highly conservative 5 - non coding region (5 - NCR) of HCV genome. The real time RT-PCR assay on the HCV 6 seropositive samples yielded reproducible positive data. Real-time PCR quantification analysis of 66 seronegative patients demonstrated 48 positive cases for HCV genome (72.7%) and 18 cases (27.3%) were negative. The results confirmed the sensitivity and specificity of real-time RT-PCR compared to serodiagnostic ELISA technique. Among the 48 positive cases by real time PCR, 30 cases showed minimal viral load (< 10 IU/ml), 8 cases showed moderate quantity (up to 10 IU/ml) and 10 cases showed strong viral load (>10° IU/ml). Furthermore, the cases with low viral load were selected for qualitative HCV detection by conventional RT-PCR and agarose gel electrophoresis. The results showed 4 (13.3%) negative cases and 26 (86.7%) positive cases. These results also reassured the importance of real-time PCR technology for HCV-detection compared to the conventional RT-PCR and highlighted the validity of applying real time PCR for the accurate diagnosis of the viral load, hence monitoring the efficiency of therapeutic drugs in hepatitis patients.