In order to fully characterize interfacial systems at the smallest scales, advanced analytical surface techniques have to be employed to render a complete picture of molecular assemblies. In this study, we carried out ultrahigh vacuum (UHV) scanning tunneling microscopy (STM) experiments on subphthalocyanine (SubPc) molecules, which are self-assembled on a Ag(100) substrate. The UHV STM experiments were complemented by tip-enhanced Raman spectroscopy (TERS), surface-enhanced Raman spectroscopy (SERS), and density functional theory (DFT) calculations. The TERS spectrum shows a high signal intensity (>600 ADU·mW–1·s–1) due to piezo-driven in-vacuo excitation and collection lenses with large numerical apertures (NA = 0.4). A new two-dimensional molecular superstructure of SubPc was discovered to consist of two distinct molecular binding configurations, both of which interact relatively weakly with the underlying metallic substrate as revealed by high-signal-to-noise enhanced Raman spectra. Our results demonstrate the necessity of advanced Raman techniques such as TERS when precisely probing molecule–molecule and molecule–substrate interactions.