The solution-based self-assembly of native and permethylated cyclodextrins (CD) bearing an azobenzene substituent has been studied by electrospray ionization mass spectrometry (ESI-MS). The results revealed that the CD molecules form either a contact or a face-to-face inclusion complex depending on the interaction of their substituents. The mass spectrometric study further demonstrated that the inclusion complex is formed through the interaction between the host CD cavity and the guest-substituent and that a contact complex is formed by hydrogen-bonding of the hydroxyl functions at the rims of the CD molecule. We also found that in order to detect the face-to-face inclusion complex by ESI-MS, the following conditions have to be met: (1) The CD moieties must be permethylated to avoid formation of the contact complex, (2) they must possess a guest-substituent of suitable length, such as an azobenzene moiety, and (3) they must possess an NH(2) or OH group at the substituent terminals for protonation and for detection as cations by ESI-MS. Formation of the inclusion complexes was further confirmed by the synthesis of a capped inclusion dimer and a capped monomer. Collision-induced dissociation (CID) experiments have been carried out for the contact, the host-guest inclusion, and the capped inclusion dimers, and the contact complexes are found to be the most stable among them.