We report on an experimental procedure in confocal single molecule fluorescence lifetime correlation spectroscopy (FLCS) to determine the range of excitation power and molecular or particulate concentration in solution under which the application of an unmodified model autocorrelation function is justified. This procedure enables fitting of the autocorrelation to an accurate model to measure diffusion length (r) and diffusion time (τ_D) of single molecules in solution. We also report on the pinhole size dependency of r and τ_D in a confocal FLCS platform. This procedure determines a set of experimental parameters with which the Stokes–Einstein (S–E) equation accurately measures the hydrodynamic radii of spherical nanoparticles, enabling the determination of the particle size range for which the hydrodynamic radius by the S–E equation measures the real particle radius.