Although data on the behavior of tocopheryl acetate (Tac) in the gut is rare, some studies show that this ester is hydrolyzed in the intestine and the released tocopherol (Tol) is absorbed. An HPLC method is published for the simultaneous analysis of Tol and Tac in mixed micelles. This study shows how this method is used for the measurement of esterase activity on Tac using commercial cholesterol esterase. The rate of hydrolysis is determined in less than 20 min on the basis of both Tac disappearance and Tol appearance. The present method is useful for investigating esterase activities on Tac in model systems that mimick the absorption environment or in any biological or nonbiological medium.