The current study focuses on the interaction between hierarchical structures of nanoparticles (NPs) and a semiconductor substrate on which they are assembled. Monolayer and bilayer assemblies of two different NPs were prepared on the surface of a GaAs substrate. The photoluminescence response of the bilayer assemblies depends on their hierarchy, namely on the ordering of differently sized nanoparticles with respect to the surface; however, the surface photovoltage does not. Based on these studies, it is possible to determine the importance of each of the possible quenching mechanisms for electron-hole pair excitation.