(1) Background: Several members of the ubiquitous aquaporin family, AQP, of water and neutral solute channels carry a cysteine residue in the selectivity filter region. Traditionally, toxic mercury-containing compounds are used to bind to the cysteine as covalent AQP inhibitors for physiological studies or analysis of structure–function relationships. (2) Methods: We tested thiol-reactive methylthiosulfonate reagents, MTS, as alternative Cys modifiers for AQP inhibition. Three MTS reagents transferring S-alkyl moieties of increasing size, i.e., S-methyl, S-n-propyl, and S-benzyl, were used with yeast-expressed water-selective AQP1 and the aquaglyceroporin AQP9. Respective Cys-to-Ala variants and mouse erythrocytes that naturally express AQP1 and AQP9 served as controls. (3) Results: Both wildtype AQP isoforms were inhibited by the Cys modifiers in a size-dependent manner, whereas the Cys-to-Ala-variants exhibited resistance. Sub-millimolar concentrations and incubation times in the minute range were sufficient. The modifications were reversible by treatment with the thiol reagents acetylcysteine, ACC, and dithiothreitol, DTT. (4) Conclusions: MTS reagents represent a valid alternative of low toxicity for the inhibition of mercurial-sensitive AQPs.