A simple square wave voltammetric method for trypsin activity determination, based on 1,2-benzoquinone electrochemical reduction on gelatin coated screen printed electrodes requiring small (30 mu L) sample volume was developed. The proteolytic digestion of the gelatin film facilitated the transport of the electroactive specie to the electrode surface and resulted in reduction peak current increase. Current response as a function of trypsin concentration within 10 min of incubation time was evaluated in the range of 0.1-1000 mu g mL(-1) trypsin corresponding to enzyme activity in the range from 0.75 U mL(-1) to 7500 U mL(-1), respectively. The limit of detection was determined to be as low as 0.01 mu g mL(-1) (0.075 U mL(-1)) trypsin after 140 min incubation time. The optimal hydrogel permeability defined by the concentration and the volume of the deposited gelatin solution (6% and 8 mu L, respectively) was established performing preliminary experiments using a gelatin modified glassy carbon electrode of conventional type and applying factorial design approach. The method allows a rapid trypsin activity determination in the normal and acute pancreatitis range, taking advantage of the low cost and disposability of the mass produced screen printed electrodes, suitable for point-of-care testing. (C) 2012 Elsevier Ltd. All rights reserved.