This manuscript analyzes the dynamics of coalescence of an incoming aqueous plug with a wetting layer above a hydrophilic surface in the chemistrode. The chemistrode is a recently described (Chen, D.; Du, W.; Liu, Y.; Liu, W.; Kuznetsov, A.; Mendez, F. E.; Philipson, L. H.; Ismagilov, R. F. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16843-16848) microfluidic analogue of an electrode, but operating at the chemical rather than electrical level, developed with the aim of capturing local stimulus-response processes in chemistry and biology. The chemistrode consists of open-ended V-shaped microfluidic channels that can be brought into contact with a chemical or biological hydrophilic substrate. The chemistrode relies on multiphase aqueous/fluorous flow and uses plugs to achieve high temporal resolution of stimulation and sampling. Coalescence of the incoming plugs, containing the stimuli, with the liquid in the wetting layer is required for chemical exchange to take place in the chemistrode. Here, we investigate the system with triethyleneglycol mono[1H, 1H-perfluorooctyl] ether RfOEG as the surfactant. This surfactant was necessary to prevent nonspecific absorption of proteins to the aqueous fluorous interface and to ensure biocompatibility of the system, but too much surfactant increased the barrier for coalescence. In this system, coalescence was controlled by the capillary number. At a higher value of the capillary number, coalescence took more time, and deformation of the interface of the incoming plug and the wetting layer was more significant. Above a critical capillary number, coalescence did not occur between the incoming plug and the wetting layer. The critical capillary number was an increasing function of surface tension but was independent of viscosity ratio. Coalescence was surprisingly reproducible, presumably because film rupture during coalescence was reliably initiated at the hydrophilic substrate. These results are useful in rational operation of the chemistrode and also provide an experimental description of deformation, film drainage, and coalescence of surfactant-coated droplets in an external flow field.