The synthesis of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins was achieved according to the standard protection-deprotection procedure. The formation of inclusion complexes between the amphiphilic alpha-, beta- and gamma-cyclodextrins and an antiviral molecule, acyclovir (ACV) was investigated by UV-visible spectroscopy (UV-Vis) and electrospray ionisation mass spectrometry (ESIMS). UV-Vis spectroscopy allowed determination of the stoichiometry and stability constants of complexes, whereas ESIMS, a soft ionisation technique, allowed the detection of the inclusion complexes. The results showed that the non-sulfated amphiphilic cyclodextrins exhibit a 1:2 stoichiometry with acyclovir, while sulfated amphiphilic cyclodextrins, except gamma-cyclodextrin, exhibit a 1:1 stoichiometry indicating the loss of one interaction site. Non-covalent interactions between acyclovir and non-sulfated amphiphilic cyclodextrins appear to take place both in the cavity of the cyclodextrin and inside the hydrophobic zone generated by alkanoyl chains. In contrast, in the case of sulfated amphiphilic cyclodextrins, the interactions appear to involve only the hydrophobic region of the alkanoyl chains.