Buffer liquids currently used in biotechnology contain surfactants. These surfactants modify the wetting properties of the liquid and affect the behavior of droplets in electrowetting on dielectric (EWOD) microsystems. An analysis of the effect of surfactants in EWOD-based microsystems, at equilibrium and in a non-equilibrium state, is given here. First, a reference for the value of the surface tension and its time variation is obtained by the use of the pendant drop method, showing that the time variation of the surface tension follows a decreasing exponential law. Next, equilibrium experiments have been performed on a specially designed EWOD experimental microsystem. It has been observed that the Lippmann–Young theoretical equation for electrowetting is satisfied at equilibrium, even in the presence of surfactants. However, at the CMC, equilibrium surface tension values are different from those obtained by the pendant drop method. This difference is explained by an internal convective motion that remixes the surfactants. This internal motion is observed by following the motion of fluorophores. Consequently, surfactant concentrations about ten times the CMC are required to obtain the same values of the surface tension in the pendant drop method and by EWOD measurements. In a second section, it is shown that the behavior of surfactant-loaded droplets in EWOD-based microsystems can be explained by considering that the surface tension relaxes with time according to the exponential law found in the first section.