Optical second harmonic generation (SHG) has a high potential for studying surface properties of colloidal particles in situ with a high temporal resolution. In the past the SHG technique has been applied successfully to determine the surface potential of colloidal particles and emulsion droplets and to study adsorption processes at the interface of solid and liquid particles. However, in previous studies mainly monodisperse particles were used. Here we demonstrate that it is also possible to study adsorption processes on polydisperse particle systems with SHG by comparing in situ adsorption measurements on monodisperse and on polydisperse mixtures of polystyrene (PS) particles. In addition, the results suggest that SHG is also applicable for surface characterization of polydisperse samples at high particle concentrations, where absorption and scattering of the particles is dominant.