A successful hyphenation of in-tube solid phase microextraction (SPME) and pressure-assisted capillary electrochromatography (pCEC) was developed by installing a poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary to the six-port valve in CEC system.The device designed was appropriate for online in-tube SPME coupled to pCEC or capillary high performance liquid chromatography (μHPLC).The evaluation of this hyphenation was first carried out for in-tube SPME-μtHPLC with analytical capillaries packed with 3 μm octadecylsilica (ODS).Theobromine, theophylline and caffeine were chosen as model drugs for an easy comparison with the results obtain by in-tube SPME-HPLC.The detection limits of these three analytes were improved more than 100 times when compared with the direct analysis by μtHPLC.Then in tube SPME-pCEC with CEC capillaries packed with perphenylcarbamoylated β-cyclodextrin bonded-silica particles was applied to the determination and analysis of propranolol enantiomers in human urine.Under optimal extraction and separation conditions, the experimental limits of detection were 4 and 7 ng/mL for (S)-propranolol and (R)-propranolol, respectively.The calibration curves showed good linearity for both (S)-propranolol (R2=0.9997) and (R)-propranolol (R2=0.9996) over the concentration range from 20 to 5000 ng/mL.Reproducibility of the method was also investigated with intra-and interday precisions lower than 10% for both enantiomers at different concentration levels.