Hepatitis C virus (HCV) has been estimated to affect around 170 million people worldwide. The cellular innate immune response recognizes HCV through pattern recognition receptors (PRRs), resulting in the production of Type I and III interferons (IFNs) that in turn drives interferon stimulated gene (ISG) expression, many of which are antiviral. However, HCV can subvert innate immune recognition at multiple levels, which is thought in part to be responsible for disease progression and chronic infection. While many studies have investigated the cellular transcriptome to viral infection at the bulk cell level, investigation of the single cell could inform us of hierarchical responses to viral infection and the innate immune response in infected and non-infected bystander cells. Therefore, transcriptome analysis at the single cell level is necessary to further our understanding of the dynamics of the cellular response to HCV infection. Here we describe and validate a methodology for single cell analysis of ISG expression by RT-PCR in the context of interferon-α stimulation and HCV replication using Fluidigm’s Biomark HD. Initial studies revealed considerable heterogeneity in ISG expression in IFN-α treated Huh-7 cells. Further analysis involving the comparison of gene expression profiles between HCV infected and bystander cells following co-culture of IFN-α treated Huh-7.5 cells harboring a HCV sub-genomic replicon (SGR) with unstimulated Huh-7.5 parent cells revealed segregation and heterogeneity within transcriptional expression between these distinct subpopulations of cells. As anticipated, this can be related to the presence of HCV replication and/or exposure to IFN-α. Ongoing studies of IFN and HCV-induced changes in gene expression profiles at the single cell level in the context of primary hepatocyte cultures and infected liver tissue may reveal gene expression signatures that are relevant to effective clearance of HCV infection by host innate immune responses.