A sensitive HPLC method for the quantification of rogletimide enantiomers in serum with UV detection was described. The method involves the use of a solid phase extraction of the R(+) and S (−) enantiomers of rogletimide and the internal standard S(−) amino glutethimide from serum using a C18 Bond-Elute column. Chromatographic resolution of the enantiomers was performed on a reversed phase cellulose-based chiral column (Chiralcel OJ-R) under isocratic conditions using a mobile phase consisting of 80:20 v/v 0.25 M aqueous sodium perchlorate-acetonitrile (pH 5.6 adjusted with perchloric acid) at a flow rate of 0.5 mL/min. Recoveries for R (+) and S (−) rogletimide enantiomers were in the ranges of 84–89% at 200 – 1000 ng/mL level. Intra-day and inter-day precision calculated as % RSD were in the ranges of 3–4% and 1–5% for both Intra-day and inter-day accuracies calculated as % error were in the ranges of 2–4% and 1.5–4% for both enantiomers, respectively. Linear calibration curves were in the concentration ranges of 100–1500 ng/mL for each enantiomer in serum. The limit of quantification of each enantiomer was 100 ng/mL. The detection limit for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/mL (S/N=2).