Deep eutectic solvents (DESs) are emerging molecular liquids with highly tunable supramolecular architectures, offering sustainable alternatives to volatile solvents in natural product analysis. However, their complex composition and strong hydrogen-bond networks can interfere with metal-based spectrophotometric assays, such as the aluminum chloride method widely used for flavonoid determination. This study explores the molecular interference effects of structurally diverse DESs—composed of choline chloride and various hydrogen bond donors—on Al3+–flavonoid complexation using rutin as a model analyte. Spectroscopic data revealed strong suppression of absorbance in the presence of acid-based DESs, with up to 98 % signal loss due to competitive coordination with aluminum ions. Quantum chemical calculations demonstrated that DES components form energetically favorable hydrogen-bonded adducts with rutin, further perturbing complex formation. To overcome this interference, a metal-free, pH-adjusted method was developed, promoting the formation of stable flavonoid chromophores at pH 10. The method exhibited high sensitivity (LOD = 1.03 mg L−1), precision (RSD < 2.2 %), and environmental compliance (AGREEprep score: 0.78). Validation in DES-containing extracts of Sanguisorba officinalis confirmed its robustness. This work provides mechanistic insight into solvent–analyte–reagent interactions in DES-rich systems and establishes a sustainable analytical approach for flavonoid quantification in complex molecular liquid media.