Polyrotaxanes are supramolecular assemblies composed of polymers, threaded macrocycles, and bulky stopper molecules to inhibit the decomposition of the first two. Cyclodextrin (CD)-based polyrotaxanes are promising therapeutic agents for lysosomal storage disorders, as they can transport CDs into the cells to normalize the intracellular trafficking of lipids, such as glycolipids or even cholesterol. Even though the important application of these supramolecular polymeric assemblies, their application is highly hindered by the costly, multistep synthesis methods, as well as the low aqueous solubilities of the non-modified polyrotaxanes. We have developed a simple one-pot synthesis of CD-based polyrotaxanes with poly(ethylene glycol) (PEG) and poly(e-caprolactone) (PCL) axes using glutathione-sensitive disulfide-connected stopper molecules. This new synthetic pathway provides high threading efficacy and, due to the versatility of threaded CDs, also tunable water solubilities. In the case of PCl polyrotaxanes, high biodegradability was also detected, using lipase as a model enzyme. Cellular uptake studies on the Caco2 cell line showed up to 52-fold enhanced cellular internalization of these supramolecular assemblies compared to free CDs. In addition, the glutathion-triggered reductive removal of the stopper molecules showed the potential decomposition of these polyrotaxanes in target cells. Based on these results, the synthesized polyrotaxanes with disulfide stopper molecules might be promising supramolecular excipients for cellular delivery of a-CD and its derivatives.