The hydrodynamic coupling between two isolated 0.97 mu m diameter polystyrene spheres is measured by constructing their Brownian motion using digital video microscopy. Blinking optical tweezers are used to facilitate data collection by positioning the spheres in the microscope’s focal plane and in close proximity to one another. The observed separation dependence of the spheres’ relative and center of mass diffusion coefficients agree with that predicted by low Reynolds number hydrodynamics and the Stokes-Einstein relation.