High molecular weight polyrotaxanes are usually characterized by nuclear magnetic resonance and size exclusion chromatography but rarely by mass spectrometry. This article reports effort to detect high molecular weight structures of cyclodextrin based polyrotaxanes (CD based PRs) by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). A particular attention was paid to verify the composition of the polyrotaxanes by determination of the number of macrocycles borne by the polyrotaxane and the polyrotaxanes synthetic route. Various matrices including crystalline or ionic liquid ones were screened to optimize the detection. Dimethylformamide as solvent widely used in supramolecular chemistry, but unusual in mass spectrometry, was successfully employed during all analyses. To prove the ability of the optimized MALDI-TOF MS conditions, three different polyrotaxane structures varying by polymer nature, i.e., poly(ethylene oxide) or polydimethylsiloxane and cyclodextrins type i.e. alpha or gamma with or without modifications were evaluated. Finally, we have concluded that 1,1,3,3-tetramethylguanidinium salts of both 2-(4-hydroxyphenylazo)benzoic acid and 2',4',6'-trihydroxyacetophenone, are the most reliable and efficient matrices. These ones afforded the possibility to detect masses of 24804 and 26447 g.mol(-1), corresponding to poly[17]- and poly[18]rotaxanes, respectively.