<p>Anxiety disorders are prevalent psychiatric conditions that frequently emerge during adolescence. Among the neurobiological systems implicated in these disorders, the endocannabinoid (eCB) signaling system plays a crucial role, making it a promising target for therapeutic interventions. In addition to its direct effects on anxiety regulation, eCBs may also influence response to first-line pharmacologic treatments, such as selective serotonin reuptake inhibitors (SSRIs). However, little is known about developmental changes in eCB lipids—<i>N</i>-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG)—or their relationship to anxiety symptoms and treatment response. Circulating AEA and 2-AG concentrations were measured in youth (aged 9–17, <i>N</i> = 199) with varying anxiety symptoms, assessed using the Screen for Child Anxiety-Related Disorders (SCARED). We evaluated how eCBs relate to developmental factors (e.g., demographics, biological variables) and anxiety symptoms (SCARED total). Additionally, we examined how eCB concentrations change in response to acute SSRI treatment in a subsample of adolescents (age 12–17, <i>N</i> = 41) with generalized anxiety disorder (GAD), who participated in an 8-week randomized placebo-controlled trial of escitalopram (15 mg/day, titrated to 20 mg/day). Body mass index (BMI) was positively correlated with circulating AEA, while 2-AG showed negative associations with age, female sex, and time-of-day. After adjusting for these variables, more severe anxiety symptoms were associated with higher AEA and lower 2-AG. Greater increases in 2-AG from baseline (without changes in AEA) were linked to improved treatment response in adolescents with GAD. Our study suggests that circulating eCBs may serve as biomarkers for anxiety severity and predictors of treatment response in youth.</p><p>ClinicalTrials.Gov Identifier: NCT02818751.</p>