A method was developed for simultaneous preconcentration and chiral separation of thalidomide enantiomers in human urine by CEC in combination with self-concentration and solvent gradient effects. A 4 cm long octyl (C8) monolithic column was hyphenated with a 15 cm long norvancomycin (NVC)-bonded monolithic column via a fluorinated ethylene-propylene interface. Sample solution was injected into the C8 monolithic column, the two thalidomide enantiomers were first preconcentrated on the C8 monolithic column, and then separated with a further concentration on the NVC-bonded monolithic column by CEC. Injection of 34.8 mm plug of sample solution gave 278- and 298-fold enhancement in sensitivity, and detection limits of 90 and 94 mu g/L for the two thalidomide enantiomers. Peak areas of the two isomers were linear in a range of 0.5-50 mg/L. The precision for five replicate injections of 10 mg/L were 0.8-0.9 and 1.1-2.3% for the migration time and peak height, respectively. The developed method was applied to the determination of racemic thalidomide in spiked human urine samples.