We have developed a novel 96-well microtiter plate high-throughput screening filtration assay for the detection of helicase activity. In this paper we present data for detection of helicase activity of the UL5/8/52 helicase-primase complex from herpes simplex virus 1 (HSV1). The assay involves the detection of radioactively labeled oligonucleotide annealed to a single-stranded circular DNA following capture of the annealed complex on silica beads. We have screened over 200,000 samples containing small organic molecules and natural products and identified T157602, a two-amino thiazole, as a specific inhibitor of HSV replication.