Abstract: Objective To separate the enantiomers of citalopram and determine the content of S-citalopram using chiral high-performance liquid chromatography (HPLC). Methods The chiral column CHIROBIOTIC V was used with the mobile phase using methanol-acetic acid-triethylamine (100:0.1:0.1) at the detection wavelength of 240 nm, column temperature of 20℃ and flow rate of 1.0 ml/min. Results Complete separation of the enantiomers of citalopram was achieved, and S-citalopram exhibited good linearity within the concentration range of 10 to 150 μg/ml (r=0.9991, n=5). Conclusion This method allows accurate quantification of S-and R-citalopram and is well suited for drug interaction investigations.