Hepatitis C virus (HCV) infection is the leading cause of chronic liver disease that currently affects at least 170 million people worldwide. Although significant efforts have been focused on discovering inhibitors of a viral polymerase (NS5B) or protease (NS3), strategies to cure HCV infection have been hampered by the limited therapeutic target proteins. Thus, discovery of a novel target remains a major challenge. Here, we report a method that combines transcriptome expression analysis with unbiased proteome reactivity profiling to identify novel host cell response factors in HCV infection. A chemical probe for non-directed proteomic profiling was selected based on genome-wide transcriptome expression analysis after HCV infection, which revealed noticeable alterations related to disulfide bond metabolism. On the basis of this result, we screened the proteome reactivity using chemical probes containing thiol-reactive functional groups and discovered a unique labeling profile in HCV-infected cells. A subsequent quantitative chemical proteomic mapping study led to the identification of a target protein, T-plastin (PLST), and its regulation of HCV replication. Our approach demonstrates both a straightforward strategy for selecting chemical probes to discriminate disease states using a model system and its application for proteome reactivity profiling for novel biomarker discovery.