Background Pediatric mood and anxiety disorders are common and associated with significant morbidity and mortality, including suicidality. Understanding factors influencing symptom presentation and treatment response will aid in developing targeted therapies and optimizing outcomes. Neurochemical levels serve as proxies for neurobiological processes altered in depressive and anxiety disorders. While the impact of antidepressant treatment on brain metabolism has been studied in adults, there are limited data in pediatric populations. This study aims to evaluate the relationship between neurochemical concentrations and anxiety and depressive symptoms in fluoxetine-treated youth. Methods Adolescents and young adults aged 12-21 years, on steady-state fluoxetine, underwent proton magnetic resonance spectroscopy (1H-MRS) at 3T to measure glutamate (Glu), glutamine (Gln), myo-inositol (mI), choline-containing products (Cho), N-acetyl-aspartate (NAA) and creatine (Cr). Neurochemicals were reported as ratios relative to Cr. Depressive and anxiety symptoms were assessed using the PHQ-9 and PROMIS Anxiety scales, respectively. Participants with PHQ-9 scores ≥ 11 or PROMIS Anxiety t-scores ≥ 60 (moderate anxiety) were considered non-responders. Statistical analyses included Student’s t-test, Cohen’s d, and Spearman’s rho, performed using JMP Pro v17.  Results In 46 youth (mean age 16.1 ± 1.9 years, range 12-21, 67% female), NAA was significantly higher in fluoxetine responders compared to non-responders (NAA mean 1.67 ± 0.09 vs 1.57 ± 0.11, p = 0.004, Cohen’s d = 0.94) when stratified by response to fluoxetine. NAA levels did not correlate with anxiety symptom severity (Spearman’s ρ= -0.146, p=0.333). Glx, Glu, Gln, mI, and Cho levels did not differ between responders and non-responders, nor did they correlate with anxiety or depression symptom severity scores. Conclusion In fluoxetine-treated youth, fluoxetine anxiety responders had higher NAA levels compared to non-responders. NAA, a marker of neuronal integrity, is typically lower in certain psychiatric disorders relative to healthy controls, with some evidence suggesting normalization following successful treatment. Baseline levels of NAA were not measured to provide insight on whether the fluoxetine responders had higher NAA levels before starting treatment. Our pilot findings support the need for further research into NAA as a potential biomarker for fluoxetine response.