A method for measuring the pair interaction potential between colloidal particles by extrapolation measurement of collective structure to infinite dilution is presented and explored using simulation and experiment. The method is particularly well suited to systems in which the colloid is fluorescent and refractive index matched with the solvent. The method involves characterizing the potential of mean force between colloidal particles in suspension by measurement of the radial distribution function using 3D direct visualization. The potentials of mean force are extrapolated to infinite dilution to yield an estimate of the pair interaction potential, U (r). We use Monte Carlo (MC) simulation to test and establish our methodology as well as to explore the effects of polydispersity on the accuracy. We use poly-12-hydroxystearic acid-stabilized poly(methyl methacrylate) (PHSA-PMMA) particles dispersed in the solvent dioctyl phthalate (DOP) to test the method and assess its accuracy for three different repulsive systems for which the range has been manipulated by addition of electrolyte.1