Two high-performance liquid chromatographic methods for the determination of enantiomers of E2020, 1-benzyl-4-[(5,6-dimethoxy-1-indanon)2-yl]methylpiperidine (I) hydrochloride, in rat plasma have been developed. The first method involves chiral separation of I on an ovomucoid-bonded column, and native fluorescence detection of I with excitation at 318 nm and emission at 390 nm. The fluorometric detection is without interference from background components and is about five times more sensitive than ultraviolet detection at 271 nm. The method was successfully applied to monitoring the racemization of each enantiomer of I in buffer solutions and in rat plasma. The second method involves separation of I from background components of rat plasma on an achiral column, collection of the I fraction into a sample loop, concentration or I to a trap column, transfer of I to a chiral column, resolution of the enantiomers of I on the chiral column and fluorometric detection of the enantiomers of I with excitation at 318 nm and emission at 390 nm. The detection limits of I and each enantiomer of I were 1 and 1 ng/ml, respectively, with a 200-microliters injection of deproteinized plasma samples.