A small molecule library containing 480 known bioactive compounds was screened for antiviral activity against poliovirus (PV) using a cellular fluorescence resonance energy transfer (FRET) assay for viral protease activity. The infected reporter cells treated with the viral replication-suppressing compounds were examined via fluorescence microscope 7.5 h postinfection. Twelve molecules showed moderate to potent antiviral activity at concentrations less than 32 microM during the primary screening. Three compounds, anisomycin, linoleic acid, and lycorine, were chosen for validation. A dose-dependent cytotoxicity assay and a secondary screening using conventional plaque assay were conducted to confirm the results. The developed method can be used for rapid screening for molecules with antiviral activity.