The human polyomavirus JC virus (JCV) is responsible for the CNS demyelination observed in cases of progressive multifocal leukoencephalopathy. The JCV regulatory region (promoter) is a hypervariable, noncoding, nucleotide sequence positioned between the early and late protein-coding regions in the viral genome. Selective binding of cellular transcription factors to this promoter region participates in the control of viral tropism. Hence, further study of these proteins might provide new insights into JCV tropism and associated pathogenesis. This review gives an overview of viral proteomics - the study of all proteins expressed from the viral gene transcripts, and all the cellular proteins that play a role in JCV tropism. It also describes a new biochemical approach for studying relevant JCV promoter-binding proteins, which is an anchored-JCV transcriptional promoter (ATP) assay. An ATP assay utilizes the product of PCR-amplified JCV promoter sequences coupled with Sepharose beads in order to capture and isolate cellular nuclear proteins with specific promoter-binding affinity for analysis. Proteins that bind to JCV-ATPs can be eluted and subjected to proteomic analysis. Insights from this approach may improve the understanding of viral and cellular parameters that control JCV tropism.