Abstract Background: Hepatitis C virus (HCV) infection is now becoming a common health problem in both developed and developing countries. The limitation of the available diagnostic approaches enhances the efforts to have a rapid, sensitive, and specific diagnostic testing for HCV infection. Capillary zone electrophoresis (CZE) is a fully automated analytical technique whose popularity is quickly increasing in the clinical chemistry laboratory. CZE can analyze nanoliters or less of samples with detection sensitivity at the attomole level (10 −18 mol) or less. Methods: CZE was optimized for the identification of a specific marker of HCV infection. The performance characteristics of the CZE for the detection of HCV RNA peak were evaluated in comparison with standard nested PCR. Results: A characteristic peak at 2.72 min was identified only in the CZE electropherogram of urine samples from HCV-infected individuals. The nature of the characteristic peak was investigated and confirmed to be HCV RNA using PCR and other biochemical treatments including RNase, DNase, and trypsin enzymes. CZE showed high degrees of sensitivity (94%) and specificity (96%) in comparison with the nested PCR. Conclusion: CZE provides a rapid, inexpensive, sensitive, and specific analytical method for diagnosis and mass screening of a large number of HCV-infected individuals.