A method for the quantitation of ibuprofen enantiomers in human plasma has been developed and validated. Separation of R- and S-ibuprofen was achieved on a silica-bonded beta-cyclodextrin column with a mobile phase of acetonitrile-0.02% (v/v) triethylamine in water adjusted to pH 4.0 with glacial acetic acid in water (60:40, v/v). The UV detection was performed at 220 nm. The established linearity range was 1-25 micrograms ml-1 (r > 0.99). The limit of quantitation was designed as 1 microgram ml-1 for each enantiomer. Interday precision and accuracy for the standards were 2.2-5.9% relative standard deviation (RSD) and -2.9(-)+3.5% relative error for R-ibuprofen, and 1.9-6.3% RSD and -7.1(-)+4.4% relative error for S-ibuprofen. Interday precision and accuracy for quality controls at 2.5, 7.5 and 17.5 micrograms ml-1 were 6.1-6.4% RSD and -1.4(-)+0.8% relative error for R-ibuprofen, and 5.7-5.9% RSD and -1.2(-)+2.8% relative error for S-ibuprofen. p-Isopropylbenzoic acid was used as an internal standard. The run time was 26 min. Interference from various lots of human plasma were not observed. Stability results of on-system, re-injection, bench-top, freeze-thaw cycles and sample storage were established.