The design of supramolecular networks based on organic molecules deposited on surfaces, is highly attractive for various applications. One of the remaining challenges is the expansion of monolayers to well-ordered multilayers in order to enhance the functionality and complexity of self-assemblies. In this study, we present an assessment of molecular conformation from 2D to 3D supramolecular networks adsorbed onto a HOPG surface under ambient conditions utilizing a combination of scanning probe microscopies and atomic force microscopy- infrared (AFM-IR). We have observed that the infrared (IR) spectra of the designed molecules vary from layer to layer due to the modifications in the dihedral angle between the C=O group and the neighboring phenyl ring, especially in the case of a 3D supramolecular network consisting of multiple layers of molecules.