Human cytomegalovirus (HCMV) infection is responsible for severe, often even fatal, diseases in immunocompromised subjects and also represents the major cause of viral-associated congenital malformations in newborn children. The few drugs licensed for anti-HCMV therapy suffer from many drawbacks and none of them have been approved for the treatment of congenital infections. Furthermore, the emergence of drug-resistant viral strains represents a major concern for disease management. Thus, there is a strong need for new anti-HCMV drugs. Here we describe three different assays for the discovery of novel anti-HCMV compounds: two are in vitro assays, i.e., a fluorescence polarization (FP)-based assay and an enzyme-linked immunosorbent assay (ELISA), which are designed to search for compounds that act by disrupting the interactions between the HCMV DNA polymerase subunits, but in general can be employed to find inhibitors of any protein-protein interaction of interest; the third is a cell-based assay designed to identify inhibitors of the viral immediate-early 2 (IE2) protein activities.