Enzymology of non-aqueous media, also termed a non-conventional enzymology, is a discipline focused on chemical reactions catalysed by enzymes in the media other than water. However, such environments always contain certain amount of water, either dissolved in a solvent or enzyme-bound. The latter fraction of water is called an essential one, and it is crucial for the non-aqueous enzymology, since it stabilises the conformation of protein molecule and determines its enzymatic activity. The paper presents structure, function, and methods of assays of the enzyme-bound water together with other water forms that can also be found in reaction milieu. The methods of regulation of enzymatic activity in non-aqueous media by using compounds capable of associating with polar atoms of amino acid residues involved in catalysis, and thus positively influencing activity of enzymes, have also been discussed. The factors giving rise to a decrease in catalytic activity of enzymes under non-aqueous conditions, and methods of prevention against this phenomenon have also been analysed.