For efficient coupling of droplet-based microfluidics with mass spectrometry (MS), a spyhole drilled on the top of a microchip is used to sample the passing droplets by electrostatic-spray ionization (ESTASI) MS. The technique involves placing an electrode below the chip under the spyhole and applying high-voltage pulses. Electrospray occurs directly from the spyhole, and the droplet content is analyzed by MS without a dilution or oil removal step. To demonstrate the versatility of this technique, we have successfully monitored a droplet-based tryptic digestion, as well as a biphasic reaction between β-lactoglobulin in water and α-tocopheryl acetate in 1,2-dichloroethane, where the protein extracts the antioxidant from the oil phase and becomes reduced.