Hydrogel substrates with a stiffness gradient have been used as a surrogate of the extracellular matrix (ECM) to investigate how cells respond to the stiffness of their surrounding matrix. Various fabrication methods have been proposed to create a stiffness gradient in the hydrogel substrate, and some of them rely on generating a concentration gradient in a prepolymer solution before photo-polymerization. One easy way to do so is to coalesce two prepolymer solution drops of different stiffness values in a narrow confinement formed by two glass surfaces and then to induce polymerization using ultraviolet (UV) light irradiation, as proposed by Lo et al. [Biophys. J. 2000, 79:144–152]. We have improved their method to enable modulating the obtained stiffness gradient and characterized fabricated polyacrylamide (PAAM) gels. We controlled the coalescence and mixing duration of two prepolymer drops using the lab-built Hele-Shaw cell device and glass surfaces with a superhydrophobic barrier. Limited mixing between the drops created a concentration gradient of the gel ingredient, which was converted to a stiffness gradient by UV-based photo-polymerization. Atomic force microscopy (AFM) indentation showed that the fabricated gels had the stiffness gradient zone at the center and that the width of the zone increased with the mixing duration.