Flavonoid glucosides have been reported to be more bioavailable than their rutinoside counterparts. The aim of this study is to describe a first step in the use of alpha-L-rhamnosidases (RhaA and RhaB) from Aspergillus aculeatus as a way to produce functional beverages based on their potentially increased flavonoid bioavailability. Blackcurrant juice (BCJ), orange juice (OJ), and green tea infusion (GT) were incubated with either RhaA or RhaB at 30 degreesC for 10 h. Aliquots of controls and enzyme-treated samples were taken at different time points and analyzed by high-performance liquid chromatography-photodiode-array detector-mass spectrometry of daughter fragments (HPLC-DAD-MS-MS). Both RhaA and RhaB selectively catalyze in situ the removal of terminal rhamnosyl groups in the three beverages despite the heterogeneity of assay conditions such as different rutinosides and pH. Incubation of the three beverages with the two rhamnosidases resulted in a hyperbolic decrease in the flavonoid rutinosides (anthocyanins in BCJ, flavanones in OJ, and flavonols in GT) and a concomitant increase in their flavonoid glucoside counterparts. The time required for conversion of 50% of the rutinoside into the corresponding flavonoid glucoside ranged from 30 min (RhaB-rutin in GT) to 6 h (RhaB-delphinidin 3-rutinoside in BCJ). The results presented in this paper are a step forward in the use of enzyme-treated beverages as a source of bioavailable flavonoid glucosides.