Background <p>Flavonols are plant specialized metabolites that regulate plant growth, development, and stress responses. Due to their antioxidant activity, they also confer nutritional benefits to human health. Quantification of flavonols in plant tissues typically relies on chromatographic methods such as HPLC or LC-MS, or on microscopy-based approaches using diphenylboric acid 2-aminoethyl ester (DPBA) staining to visualize flavonols in plant tissues. These methods are time-consuming and resource intensive. Here, we present a rapid, high-throughput, fluorescence-based microplate reader assay for flavonol quantification, in which the flavonol-specific dye DPBA is added to plant extracts to form fluorescent complexes.</p> Results <p>The assay was optimized for extraction efficiency and validated for sensitivity, accuracy, and reproducibility. It also shows consistency with HPLC measurements. We demonstrate its utility by quantifying flavonol levels in different tomato tissues, across different cultivars, and even between plant species. Our assay showed that reproductive tissue in tomato plants has higher flavonol levels than vegetative tissue. Also, we found variation in flavonol levels in tomato fruit skin across different laboratory and commercial cultivars, suggesting that our approach shows promise for use in genome-wide association studies to identify genetic factors underlying variation in flavonol levels. Lastly, we measured flavonol levels in dry leaves of different plants used for brewed beverages.</p> Conclusion <p>In conclusion, the assay represents a simple, robust, and scalable flavonol screening tool for studies in plant metabolism, environmental physiology, breeding, and metabolic engineering.</p>