The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.