A high-performance liquid chromatographic method was developed for the chiral separation of a new selective phosphodiesterase 5 inhibitors, tadalafil and its three isomers. The chiral separation was performed on a Chiralpak AD column. The mobile phase was hexane-isopropyl alcohol (1:1, v/v). UV detection was at 220 nm. Baseline chiral separation for the four isomers was obtained within 30 min. Each of the resolutions of the two pairs enantiomers were more than 2.0. The limits of quantitation were 0.60, 0.90, 1.20, and 1.80 ng for (6R, 12aS), (6R, 12aR), (6S, 12aS), and (6S, 12aR) isomers, respectively. Relative standard deviation of the method was below 2% (n=5). The method is suitable in quality control.