Human rhinoviruses (HRV) are the leading cause of the common cold, asthma, and chronic obstructive pulmonary disease exacerbations. This has led to the search for therapies that inhibit the HRV-induced inflammatory response or prevent viral infectivity. There is significant genetic diversity in the HRV family hence a lack of natural immunity, which also hampers the development of vaccines; HRV genetic diversity also is the reason there are no available broad-spectrum antivirals with direct action on HRV. A putative strategy is the identification of rhinovirus replication inhibitors targeting the polymerase activity. An initial step in HRV replication is the conjugation of viral peptide VPg uridine nucleotides to substrates, which serve as the primer for polymerisation. An antibody was raised against virus HRV-A16 peptide VPg N-terminal (GPYSGEPKPK) in the di-uridylylated form. The HRVA16 di-uridylylated antibody was crucial. It has been possible to reconstitute the uridylylation reaction to allow high-throughput screening and use the HRV-A16 VPg di-uridylylated antibody in the assay to identify novel inhibitors of human rhinovirus (HRV) VPg uridylylation with potential uses in the future as clinical antiviral drugs.