The rapid iterative negative geotaxis (RING) assay is a commonly used behavioral test in Drosophila based on digital imaging of flies and requires minimal equipment and space. Larval mobility can be used to reliably determine the effect of drugs on locomotion in Drosophila larvae. The effects of Valeriana officinalis extract (VoE) and melatonin (ME) were tested in Drosophila melanogaster flies and larvae. In the RING assay, the locomotor activity of flies was reduced to 30.6, 72 and 70.4% when exposed to 10 mg/ml VoE, 8 mM ME, and a combination of 8 mM ME + 10 mg/ml VoE, respectively. The performance index (PI) was reduced to 68.9% (P = 0.0058), 33.3% (P = 0.0001), and 70% (P = 0.0013) when flies were exposed to 8 mM ME, 10 mg/ml VoE, and a combination of 8 mM ME + 10 mg/ml VoE, respectively. The number of body wall contractions of Drosophila larvae (nBWC) was reduced to 47.8, 37.7 and 38.5% when larvae were exposed to 8 mM ME, 10 mg/ml VoE, and a combination of 8 mM ME + 10 mg/ml VoE, respectively. The displacement velocity (V 0 ) of larvae decreased significantly to 67.6, 60.1 and 55.6% when larvae were exposed to 8 mM ME, 10 mg/ml VoE, and a combination of 10 mg/ml VoE + 8 mM ME, respectively (P < 0.05). The effects of the VoE extract were not synergistic with ME as demonstrated by the RING assay, nBWC, and V 0 . Based on these results, valerian root extracts have demonstrated sedative effects that are better than those of melatonin in fruit flies, but did not show any increase in sedative effect when combined with melatonin.