The chemistry of silica-based materials, including zeolites, is strongly influenced by the nature and number of their silanols. In zeolites, they are either isolated (non H-bonded) silanol species, or connected (H-bonded) nests corresponding to hydrolyzed Si–O–Si bonds; their hydrophilic nature will in turn impact the overall properties of the corresponding material. It is therefore important to quantify and assess the nature of the silanol species. This work pools spectroscopic techniques to that effect. The zeolites with a very relevant MFI type structure were prepared as (i) pure siliceous silicalite-1, (ii) aluminium-containing ZSM-5 (Si/Al = 110), and (iii) molybdenum-containing silicalite-1 providing a rich spectrum of chemical compositions. The occurrence of both silanol types in silicalite-1 is confirmed as well as their removal by isomorphous substitution with molybdenum. The effect of the silanol species configuration in the presence of strong Brønsted acid sites is highlighted on ZSM-5. General guidelines for the evaluation of the silanol species in zeolites using spectroscopic approaches (IR, Raman and NMR) is proposed.