During enzymatic esterification in non-conventional media, the activity and enantioselectivity of the enzyme is significantly influenced by the water content of the reaction medium, which continuously changes as water is produced during the esterification. To provide constant reaction parameters, water activity should be kept constant. The commonly used salt hydrate pairs may be difficult to apply and often hinder enzyme activity. During the enantioselective esterification of racemic 2-bromopropionic acid in various solvents (organic solvents, ionic liquids), it was proven that the conditions related to the optimal water content required for kinetic examinations can be provided without using any salt or salt hydrate pairs. This conclusion is based on the realization that the optimal water activity can be set by first determining the initial water content that is necessary to achieve the maximum reaction rate in the given solvent.