For the rational design of processes using immobilized enzymes a mechanistic kinetic model is required, which accounts for all kinetic and thermodynamic phenomena, including the enzyme reaction, the mass transfer of the reactants between both phases, and their diffusion inside the immobilizate. For the example of enzymes immobilized in hydrogel beads suspended in an organic solvent, such a mechanistic kinetic model was obtained by a model-based experimental analysis approach. It was proven that the usually applied concentration measurements in the bulk phase are not sufficient to draw mechanistic conclusions. The most suitable measurement technique was found to be the quantification of the concentration along the radius of the hydrogel bead. These line scans, achieved by two-photon laser scanning microscopy, for the first time allowed to estimate intrinsic reaction and mass transfer parameters simultaneously. Thus, the obtained intrinsic parameter estimates for the biphasic hydrogel system could be directly compared with those obtained in individual systems. This comparison revealed for the first time that the enzyme reaction was not significantly affected by the mild hydrogel encapsulation. However, a significant impact on the transport parameters was found that underlines the need for analyzing the real reaction system using mechanistic models. (C) 2010 Elsevier Ltd. All rights reserved.