An electrokinetic driven microfluidic lab-on-a-chip was developed for glucose quantification using double-enzyme assay. The enzymatic glucose assay involves the two-step oxidation of glucose, which was catalyzed by hexokinase and glucose-6-phosphate dehydrogenase, with the concomitant reduction of NADP(+) to NADPH. A fluorescence microscopy setup was used to monitor the different processes (fluid flow and enzymatic reaction) in the microfluidic chip. A two-dimensional finite element model was applied to understand the different aspects of design and to improve the performance of the device without extensive prototyping. To our knowledge this is the first work to exploit numerical simulation for understanding a multisubstrate double-enzyme on-chip assay. The assay is very complex to implement in electrokinetically driven continuous system due to the involvement of many species, which has different transport velocity. With the help of numerical simulation, the design parameters, flow rate, enzyme concentration, and reactor length, were optimized. The results from the simulation were in close agreement with the experimental results. A linear relation exists for glucose concentrations from 0.01 to 0.10 g l(-1). The reaction time and the amount of enzymes required were drastically reduced compared to off-chip microplate analysis.