We study the influence of different experimental parameters on the interaction between the probe and the sample in Kelvin probe force microscopy (KPFM) measurements. We provide a precise and reproducible determination of the local surface potential (SP) of clean macroscopic highly oriented pyrolytic graphite (HOPG) samples and of organic semiconducting nanostructures of an alkyl-substituted perylene-bis(dicarboximide) (PDI) self-assembled at surfaces. We distinguish two different terms in the measured SP, intrinsic and extrinsic, containing the electrical properties of the studied object and the experimental artifacts, respectively. We investigate the effect of the most relevant experimental parameters including tip−sample distance, relative humidity (RH), and potential applied to the tip, which govern the extrinsic term of the measured SP. Moreover, we devise a theoretical description of the tip−sample …