Introduction: Barberry (Berberis vulgaris L., Berberidaceae) is a well known medicinal plant with a long history of medicinal and nutritional use in Europe, Asia and America. It has various therapeutic effects, mostly related to its isoquinoline alkaloids, particularly berberine. Objectives: The aim of this study was to examine the efficiency of ultrasound-assisted extraction of berberine from root bark of Berberis vulgaris using glycerol a non-toxic, biodegradable solvent available from renewable sources. Materials and methods: Experiment was planned using Box-Behnken design. The range of design parameters (independent variables) was: temperature (X1, 20-80C), glycerol concentration (m/m) (X2, 10- 90%) and ultrasonication power (X3, 144-720 W). Berberine content of the extracts was determined using RP-HPLC-DAD, while radical scavenging activity of the extracts (RSA) was determined spectrophotometrically, using DPPH free radical (dependent variables). Responsesurface methodology was used to find the relationship between dependent and independent variables. Results and discussion: The relationship between variables was best described by quadratic models. The selected quadratic models were highly significant (P > 0.01) with r2 values over 0.93. The most important extraction variables were temperature and glycerol content. They significantly (P > 0.05) influenced berberine concentration and RSA both as linear and quadratic terms. Furthermore, ultrasonication power had a significant (P< 0.05) effect on berberine concentration as a quadratic term, as well as in interaction with the extraction temperature. The solutions to the quadratic equations have shown that the best conditions for berberine extractions are (X1, X2, X3) are 80 C, 50 % glycerol and 144 W, while the extracts showing best RSA were obtained using 80 C, 40 % glycerol and 720 W. The experimental data has confirmed these findings with deviation 5%. Conclusions: The results demonstrate that ultrasonication-assisted extraction using glycerol is a fast and efficient method for preparation of extracts with notable berberine concentration and radical scavenging activity.