<p>Tramadol, a widely used opioid analgesic, has been linked to neuropsychiatric side effects, including anxiety, when used chronically. These effects are believed to arise from disruptions in multiple neurotransmitter systems, including GABA, glutamate, dopamine, and serotonin. Valproic Acid (VAL), known for its mood-stabilizing and neuroprotective properties, may have the potential to counteract these effects. This study aimed to investigate the behavioral and neurochemical changes induced by chronic tramadol administration in rats and to evaluate the therapeutic potential of VAL in reversing these effects. Twenty-four male Sprague Dawley rats were divided into four groups: Control, Tramadol (TRA), Valproic Acid (VAL), and Tramadol + Valproic Acid (TRA + VAL). Behavioral assessments were conducted using the Open Field Test (OFT) and Light/Dark Box (LDT). After testing, levels of GABA, glutamate, dopamine, serotonin, acetylcholine, and norepinephrine were measured in the hypothalamus and cerebral cortex using LC-MS/MS. Chronic tramadol treatment led to anxiety-like behaviors, as seen in reduced center time in the OFT and shorter latency to enter dark areas in the LDT. These behavioral disruptions were accompanied by decreased levels of GABA in the hypothalamus and cerebral cortex. Co-treatment with VAL restored GABA levels and normalized behavior. The levels of most other neurotransmitters were also affected by tramadol, but not normalized by valproate. VAL mitigates tramadol-induced neurobehavioral disturbances by restoring key neurotransmitter imbalances in GABA. These findings support the therapeutic potential of VAL in managing opioid-induced mood and behavioral disruptions in opioid use disorder.</p>