Here, we report an approach to use infrared reflection absorption spectroscopy (IRRAS) for the unambiguous proof of the presence as well as the spatial distribution of organic ligands on the Janus gold nanoparticle (AuNP) surface. For this purpose we synthesized amphiphilic and zwitterionic Janus AuNPs and immobilized these on pretreated gold surfaces by directed self-assembly, exploiting hydrophilic/hydrophobic or electrostatic interactions, respectively. Thus, we obtained macroscopic two-dimensional arrays of Janus AuNPs exhibiting a specific orientation. These arrays were investigated by IRRAS, and the obtained spectra revealed only peaks of the ligands facing the IR beam, while the ligands facing the gold substrate were not detected due to reflection of the IR beam on the AuNP cores. Thus, we describe a straightforward spectroscopic procedure to prove the Janus character of zwitterionic and amphiphilic AuNPs in the size range of 10-15 nm.