Flavonoids, a group of naturally occurring polyphenols, attract great interest due to their many apparent health benefits including their anti-oxidant properties and anti-amyloidogenic properties. However, the behavior of flavonoids in aqueous environments can be unpredictable due to the potential formation of aggregates and hydrolysis products. A better understanding of the complex behavior of flavonoids is needed before flavonoid-based therapies can be developed. Employing excitation–emission matrix (EEM) fluorescence in combination with weighted-parallel factor analysis (WPARAFAC), a series of aqueous quercetin solutions were characterized at varying pH values and concentrations. Distinct pH and concentration-dependent fluorophores were observed with emission maxima of 540 and 600 nm, respectively. However, the excitation maxima of the two species were broad and highly overlapped ranging from 350 to 400 nm. In addition, the fluorescence of quercetin was characterized in the presence and absence of bovine serum albumin (BSA). Interaction with BSA resulted in a dramatic red-shift of the excitation maximum to 445 nm, while the emission maximum of the BSA–quercetin complex was slightly blue-shifted to 535 nm. Coupling of WPARAFAC and EEM fluorescence enabled simultaneous detection of multiple fluorescent quercetin species in solution at a wide range of conditions. Copyright © 2010 John Wiley & Sons, Ltd.