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Simple Summary In our study, we looked at a behavior called prepulse inhibition (PPI) in Drosophila melanogaster, commonly known as the fruit fly. For many animals, the sudden presentation of strong sensorial stimuli can induce a defensive response or motor reflex. PPI is a phenomenon where a small stimulus, named "prepulse," is presented shortly before a larger stimulus, so the larger stimulus induces a weaker response than it normally would. This behavior is seen in many different types of animals and is used to study conditions such as anxiety and schizophrenia. For this study, the chosen stimulus is the sudden presentation of one-second darkness, or lights-off, shown to evoke an immediate locomotion response in Drosophila. Our research found that PPI can also be seen in adult flies, which has not been reported before. Additionally, we confirmed our results by showing that a drug that affects an important brain component, called the NMDA receptor, can change PPI in flies. We suggest that studying this behavior in fruit flies could help us understand how it works in other animals, including humans. Prepulse inhibition (PPI) is a widely investigated behavior to study the mechanisms of disorders such as anxiety, schizophrenia, and bipolar mania. PPI has been observed across various vertebrate and invertebrate species; however, it has not yet been reported in adult Drosophila melanogaster. In this study, we describe the first detection of PPI of visually evoked locomotor arousal in flies. To validate our findings, we demonstrate that PPI in Drosophila can be partially reverted by the N-methyl D-aspartate (NMDA) receptor antagonist MK-801, known for inducing sensorimotor gating deficits in rodent models. Additionally, we show that the visually evoked response can be inhibited by multiple stimuli presentation, which can also be affected by MK-801. Given the versatility of Drosophila as a model organism for genetic screening and analysis, our results suggest that high-throughput behavioral screenings of adult flies can become a valuable tool for investigating the mechanisms behind PPI.