Earlier, conventional methods-based cAMP-dependent PDE4B assay was found to be highly sensitive for drug screening, and the optimal working range of cAMP required to inhibit the activity of PDE4B enzyme was determined to be 0.5 µM/1 µM. Under this experimental condition, the IC50 value to inhibit PDE4B activity by roliparm and roflumilast was determined as 132 nM and 0.8 nM, respectively41,42. In the present study, a commercially available inkjet printer equipped with four cartridges was used to achieve an efficient outcome of PDE4B inhibitor activity against PDE4B enzyme in a dose-dependent manner. Gradient color codes were utilized to print gradient concentration of inhibitors per spot area. To obtain the highest sensitivity and reproducibility, the number of moles of cAMP and PDE4B were optimized using the inkjet-printing method. Our results confirm that Eu-cAMP recognizes ULight anti-cAMP in the absence of cAMP, which was confirmed by the increased FRET signal. However, cAMP remains intact and competes with the Eu-cAMP tracer for binding to the Ulight-mAb in the presence of cAMP, and thus decreases in FRET signals were recorded. Since ejection volume to be printed per spot can be controlled by adjusting the printing-output settings, the number of mole printed per spot can be evaluated. It was determined that 2.79 × 10−16 mole of cAMP successfully competes with 30 nL of Eu-cAMP tracer and ULight anti-cAMP mAb printed per spot. This optimal condition for producing the highest fluorescent intensity was selected. Similarly, three different concentrations of PDE4B were utilized to screen PDE4B activity using the inkjet printer. Here, 2.81 × 10−13 mole of PDE4B was utilized to interact with 2.79 × 10−16 mole of cAMP, where Eu-cAMP was accessible to react with ULight anti-cAMP. The maximum fluorescent signal on parchment paper was achieved in 50 min reaction time. The fluorescent signal was directly proportional to the FRET signal detected in the absence of cAMP. Our results demonstrate that FRET signals were detected on parchment paper at a lower femtomole of cAMP and picomole of PDE4B, which is proven to be more sensitive and reliable than the conventional method.