In summary, we have demonstrated a conventional inkjet-printing system for simultaneous detection of FRET signals and mole of sample consumed in the assay. It utilizes four cartridges as a generalized platform to study enzyme inhibitory activity, whereby FRET signals are generated on parchment paper. The four-cartridge printing system permits determination of mole of enzyme and inhibitor printed per spot as low as the femtomole range. Only a single sample concentration application was required for conducting the entire experiment. Given this, this paper-based enzyme inhibitory assay platform offers the major advantages of low-cost, less sample consumption, consecutive results, and fluorescent signal analysis. We expect that the sensitivity of the experiment can be further improved, and that this approach offers great potential as a novel tool for applicability of FRET determination.