Background Atopic dermatitis (AD) is an inflammatory skin disease characterized by chronic pruritic eczema with an estimated prevalence of 10% in adults, with 50% suffering moderate to severe manifestations. Dupilumab, an IL-4/IL-13 inhibitor, is approved for treating moderate to severe AD. Dupilumab-associated ocular surface disease (DAOSD) emerges in up to 60% of dupilumab-treated patients, constituting a major AD-specific adverse event. DAOSD pathogenesis has not been fully understood yet. Objective To elucidate metabolic changes occurring after dupilumab treatment in AD patients, particularly focusing on those who develop DAOSD. Methods In this prospective single-center cohort study, 20 AD patients underwent dupilumab therapy, with six developing DAOSD. Plasma and serum samples were collected at baseline, 4- and 16-weeks post-treatment initiation, and during the conjunctivitis episode. Additionally, 10 age- and sex-matched healthy controls were sampled solely at baseline. High resolution mass spectrometry was employed for metabolomic and lipidomic analysis of all blood samples. Results Targeted metabolomics and lipidomics identified 138 metabolites and lipids, while untargeted analysis revealed 7931 features in plasma or serum. Multivariate analysis unveiled significant metabolic and lipidic disparities between untreated AD patients and healthy controls, notably in AD patients who later developed DAOSD. The metabolic and lipidic profiles and their associated pathways were significantly influenced by ongoing treatment, with distinct kinetics differing between AD and DAOSD patients. Conclusion Metabolomics and lipidomics analysis further deepen our comprehension of DAOSD pathogenesis. Financial support: This project was financially supported by the City of Graz to VP and NW and the Dr. Adele-Rabensteiner Foundation of the Austrian Association of Ophthalmology to NW and by the Austrian Science Fund FWF (W1241) to PW. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. No conflicting relationship exists for any author.