"Click" chemistry, used to promote nanoparticle assemblies, is a powerful strategy which has emerged very recently to control the spatial arrangement of nanoparticles onto surfaces. Such a strategy may be of high interest for applications such as magnetic recording media or magnetic sensors which are based on the fine control of the collective properties of nanoparticles. Nevertheless, self-assembly driven by clickable functional groups still remains to be understood. Mixed self-assembled monolayers (SAMs) of alkane−thiol molecules were used to control the spatial arrangement of nanoparticles onto gold substrates. This approach was combined with click chemistry in order to control the immobilization of nanoparticles on selective areas through specific copper catalyzed alkyne−azide cycloaddition (CuAAC) reaction. Mixed SAMs consist of co-adsorbed 11-(undec-1-ynyl)thiol (S-CC) and 12-(dodecane)thiol (S-CH 3) molecules. The variation of the molar ratio between both molecules resulted in significant modulation of the structure of nanoparticle assemblies. The spatial arrangement of nanoparticles revealed the very complex structure of alkyne/methylene terminated mixed SAMs. Alkyne terminal groups could not be only studied by the usual characterization surface techniques such as PM-IRRAS and XPS. Therefore, azido-terminated nanoparticles acted as probing agents to determine the spatial distribution of alkyne groups at the surface of mixed SAMs. This approach was combined with scanning tunneling microscopy (STM) and DFT calculations to get a deeper insight into the structure of mixed SAMs of S-CC and S-CH 3 molecules. Gold substrate topography, chemical affinity of molecules, intermolecular interactions and length of alkyl chains were found to be critical parameters that rule the SAM structure.