A commercially available HP Officejet Pro 8100 printer was set to operate at normal speed. Adobe Photoshop software was utilized to attain the RGB color code, and the Microsoft Power Point tool was used to attain the desired printing patterns. Solutions of cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb were prepared with stimulation buffer containing 180 μL of PEG-400 and 100 μL of tert-butanol per 1 mL. The printing-output setting was fixed as 100, to print cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb, and buffer. The printing-output setting to define ejection volume was altered by two-orders of difference per spot (from 0 to 88) to vary the amount of roliparm or roflumilast. The time interval between printing each element (cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb, and buffer) was optimized as 10 min. Under optimized condition, printing different components layer-by-layer at the same spot created a homogeneous layer that effectively provided the mixing of assay components.