Polystyrene microtiter wells are commonly employed as substrates for immunoassays and for molecular screening. The full characterization of such substrates is therefore critical for their optimal use in such settings. In this study, various scanning force microscopy analysis modes are employed for their surface characterization. This is achieved through the spatial mapping of probe-sample interactions via lateral force microscopy, force-curve mapping, phase imaging, and pulsed force microscopy. Previous surface analytical studies had indicated the presence of a volatile hydrocarbon contaminant on well surfaces, which resulted in areas resistant to protein adsorption. These studies, however, were unable to directly determine the contaminant's dimensions or its distribution over the substrate. The obtained results confirm the presence of discrete patches of a hydrophobic material on the well surface, which additionally are found to behave as a viscous fluid. Such findings concur with the predictions made in the previous study and illustrate the potential benefits of a combined surface analytical approach.