The laminar flow regime prevailing in pressure-driven flow through a Y-shaped microfluidic channel was utilized to create a stable boundary between two aqueous liquids. Transverse transport of ions between these two liquids gave rise to a diffusion potential, which was monitored by measurement of the open circuit potential. In this report, the influence on the cross-channel potential distribution of protonation reactions occurring in the boundary zone between the two co-flowing liquids is presented. The proton source was present in one of the co-flowing streams, and an uncharged proton acceptor was present in the other aqueous stream. The time-dependent transport equation for diffusion and migration was augmented by chemical reaction terms and was solved for all species present in both streams as a theoretical basis for the analysis. Within this model, the system was assumed to be homogeneous along the channel height, and effects of nonuniform velocity profiles were neglected. A reduction in potential by several millivolts was predicted for a protonation reaction occurring close to the boundary between the two aqueous streams, provided that the mobility of the protonated species was lower than the mobility of the co-cation in the background electrolyte (alkali metal cation in this case). The magnitude of the decrease in the potential was greater for protonated molecules with lower mobility or if the mobility of the background electrolyte cation was increased. Experimental results are presented for imidazole and D-histidine as proton acceptors present in 10 mM KCl, 10 mM NaCl, or 10 mM CsCl solution and co-flowing with a stream of 10 mM hydrochloric acid, which served as the proton source. Decreases in measured potential, in line with the predicted diminished potential, were obtained.