A lipase-catalyzed enantioselective thiotransesterification between (R,S)-naproxen 2,2,2-trifluoroethyl thioester (STFs) and 4-morpholine ethanol in isooctane was employed to produce (S)-naproxen ester prodrug by controlling the initial water activity and considering the hydrolysis and esterification side-reactions. Good enzyme enantioselectivity with more than 90% of enantiomeric excess for the desired product was obtained when initial water activity and alcohol concentration were controlled at 0.113 and 10 mM, respectively. A detailed analysis of the apparent equilibrium constant and ratio of initial rates of thiotransesterification and hydrolysis varied with the water activity and alcohol concentration indicated that water and alcohol might act as modifiers in manipulating the enzyme active center aside from their conventional roles as acyl acceptor and enzyme inhibitor.