Hepatitis C virus (HCV) infection is a major global public health problem. Early induction of cross-reactive neutralizing antibodies during acute infection correlates with the spontaneous clearance of HCV. Understanding the antibody response in multiple subjects in large-scale studies would greatly benefit vaccine development. To determine the breadth of a polyclonal-serum antibody response, and or, the monoclonal antibodies against the different HCV E1E2 genotypes, we developed a quick and high throughput flow cytometry assay using fluorescent cell barcoding to distinguish cells transfected with different E1E2 sequences in a single measurement. HCV-specific antibodies recognizing conformational epitopes were tested for binding to cells transfected with E1E2 from six genotypes. In this assay, 1500 samples can be analyzed for specific binding to 6 different HCV E1E2 sequences within 8h. Plasma of HCV infected subjects were tested in our assay allowing us to determine the breadth of their antibody response. In summary, we developed a quick and high throughput assay to study the specificity of an antibody response against multiple HCV E1E2 sequences simultaneously. This assay can also be used to facilitate the discovery of novel antibodies, and because other flavi- and picornaviruses have similar intracellular assembly mechanisms, this approach can be used to study the antibody response against such viruses.