Circadian rhythm and the sleep/wake cycle can influence metabolic regulation, eating habits, hormone release, and common chronic health conditions such as obesity, depression, diabetes, and sleep disorders. the fruit fly, with its conserved molecular clocks and accessible assays, has been used as an ideal model system to study biological processes, for example, circadian rhythms, sleep, neurodevelopment, genetics, and behavior. Using an integrated approach combining high-throughput locomotor activity monitoring and untargeted metabolomics, we analyzed the behavioral and metabolic effects of a chronobiotic melatonin. The behavioral activity of fruit flies was recorded using an infrared-based monitoring device, followed by data analysis with open-source data packages ShinyR-DAM and VANESSA. We found that 1 mM and 4 mM melatonin doses significantly increased locomotor activity. Melatonin at a high concentration (4 mM) exhibited a protective effect to reduce mortality in . Despite these changes, melatonin preserved the flies' endogenous bimodal activity pattern, maintaining circadian alignment. Metabolomics analysis using high-performance liquid chromatography-mass spectrometry identified differentially abundant metabolites after melatonin administration compared with the vehicle treatment. We discovered 20 biologically relevant metabolites altered by melatonin, including key perturbations in arginine biosynthesis, alanine/aspartate/glutamate metabolism, and pyrimidine pathways. Notably, melatonin upregulated glutamine, a potential indicator of enhanced neurotransmitter synthesis and broadly modulated amino acid and nucleotide metabolism, suggesting dual roles in neuroprotection and energy homeostasis. This high-throughput omics study uncovers melatonin-induced behavioral and metabolic perturbations in as a model organism, revealing how melatonin modulates locomotor activity and circadian integrity through specific alterations in metabolism.