In a previous paper we could demonstrate the immobilization of mono- and multithiolated cyclodextrins on gold surfaces and investigated the film properties. In this paper we follow the self-assembly processes in time by optical and physicochemical methods for those three monothiolated and a mixture of multithiolated cyclodextrin derivatives. The formation of cyclodextrin films on gold substrates was found to be a multistep process involving an activation energy.