Abstract: In a method of performing dilution of a droplet in an EWOD device, a parent droplet is provided on an electrode array of the EWOD device, wherein the parent droplet has a first concentration of a species. A diluent droplet also is provided on the electrode array of the EWOD device. The method includes controlling actuation voltages applied to the electrode array of the EWOD device to join the parent droplet and the diluent droplet into a product droplet having a diluted second concentration of the species different from the first concentration in the parent droplet. The actuation voltages then are controlled to split the product droplet into one or more daughter droplets having the second concentration of the species. A dilution ratio may be calibrated based on the volumes of the droplets. Serial dilution steps may be performed to generate daughter droplets of different species concentrations at each step.