Polyrotaxanes (PR) are among the most studied interlocked molecules in the field of supramolecular chemistry. Cyclodextrin based polyrotaxanes (CD based PRs) are well-known to be difficult to analyze by mass spectrometry (MS). Nanoelectrospray (nanoESI) employed during mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments turns out to be particularly useful to analyze these noncovalent assemblies. While ESI/nanoESI based spectra usually contain multicharged species which greatly complicate the interpretation, particularly for such complex mixtures analysis, the hyphenation with a high resolution analyzer such as Orbitrap could overcome this limitation. This Article reports efforts to achieve a detailed structural deciphering by nanoESI-MS and nanoESI-MS/MS of CD based PRs constituted of αCDs, unmodified or surrounded by 1 or 2 sulfation(s), which were threaded along polydisperse poly(ethylene oxide) α,ω-dipyrenyl chains. The described method is more sensitive and less sample consuming than a typical NMR experiment and in good agreement with size-exclusion chromatography (SEC) results. Moreover, as compared to MALDI-TOF MS analysis, all populations were presumably elucidated without discrimination effect. Therefore, this MS development allowed us to estimate the PR sample content with 16 to 35 ethylene oxide units, 1 to 5 αCDs threaded, and 0 to 10 sulfo groups grafted on the overall CDs. Finally, the method afforded the possibility to unambiguously attribute supramolecular architectures from 2276.0278 to 7767.8342 g·mol(-1) corresponding to poly[2]- to poly[6]rotaxanes.