We present the fabrication, characterization and cell culture results of a microfluidic device for generating steep gradient interfaces of small molecules (<1 kDa) across cell culture with no convective shear stresses applied to the cells. We use a novel streamline of two fluids to generate stable and uniform gradient interfaces/boundaries by confronting one fluid with the other. We separate a gradient generation channel and a cell culture channel by a polyester membrane so that viscous shear stress by the bottom channel flow does not convectively disturb the chemical environment of cultured cells seeded on the membrane in the top channel. Using two-component dyes to characterize the steepness of the diffusional interface, we demonstrate 50 μm wide steps for about 400 Da molecules. Using BCECF, a 689 Da pH-sensitive diffusible dye which is actively taken up by living cells, we demonstrate gradient boundaries narrower than five cell diameters in HeLa culture. We also demonstrate steep gradients of pH across cells in the same device. This work should be of interest to researchers attempting to generate gradients of small, rapidly diffusing molecules for studies in cellular differentiation and signaling.