The fruit fly Drosophila melanogaster Meigen is a very useful model organism for studying the molecular and neural bases of nociceptive behavior. Drosophila shows robust wasabi avoidance behavior, as do mammals. Feeding-based behavioral assays have been exclusively used in previous studies of such behavior. Here, we present a novel locomotor-based behavioral assay of wasabi avoidance in Drosophila using a video tracking system. Our results showed that, in wild-type flies, the approach to a wasabi source is inhibited in a dose-dependent manner, whereas their general locomotion is unaffected. In addition, the wasabi avoidance behavior was suppressed by mutations in the painless gene, which is involved in the regulation of wasabi response. Conventional behavioral assays have not shown wasabi avoidance without ingestion, whereas our behavioral assay revealed that flies avoid highly concentrated wasabi sources even without ingestion. Thus, this behavioral assay can be used to elucidate the molecular and neural mechanisms underlying non-feeding-based wasabi avoidance in Drosophila.