BACKGROUND: Metabolomics reflects the molecular communications within biological systems. Urine is a noninvasive biofluid, rich in metabolites that serve as potential biomarkers for human health and disease. The impact of storage conditions and DNA stabilizers for urine samples in metabolomic studies remain unclear. OBJECTIVE: To evaluate the impact of common storage conditions and the presence of a DNA stabilizer, AssayAssure® (Thermo Scientific), on the metabolite content of voided human urine. METHODS: We assessed the urinary metabolite composition under different storage conditions and with the addition of AssayAssure® to determine its effect on metabolomic analysis. RESULTS: Urinary metabolite composition remained consistent across different storage conditions. However, the addition of AssayAssure® significantly altered the metabolic profile due to adduct formation. Despite these alterations, the identification of parent metabolites was not compromised, and biological differences were still distinguishable. CONCLUSION: These findings suggest that urine biobanked under the tested storage conditions is suitable for metabolomic analysis. The addition of AssayAssure® does not hinder the detection of parent metabolites, although it may affect the overall metabolic profile.