As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we have established an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. Our established experimental method included monitoring locomotor activity at high-speed movement (> 20 mm/sec), extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus. The validation study using our methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol using zebrafish is considered useful for seizure potential screening during early stages of drug discovery.