Recent advances in droplet manipulation methods by electric fields and signals require a deeper understanding of water droplet charge. In this paper, we have investigated the electrophoretic motion of individual water microdroplets injected into non-polar silicone and paraffin oil by video optical microscopy on an individual droplet basis to determine droplet charge. It was found that the initial surface charge density of surfactant free droplets directly after injection from a micropipette is positive and of the order of 10−6 C/m2, regardless of pH and ion concentration in the range from pH 4 to pH 10 and from 0.01 mmol/l to 1.5 mol/l, respectively. The experimental results together with molecular dynamics simulations show that the nature and polarity of the charge can be explained by anisotropic orientation of water molecules at the interface rather than selective adsorption of ions. Furthermore, we showed that slip at the liquid-liquid boundary must be taken into account when interpreting electrophoretic measurements of droplets.