A successful hyphenation of in-tube solid-phase microextraction (SPME) and pressure-assisted CEC (pCEC) was developed by installing a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary to the six-port valve in a CEC system. The device designed was appropriate for on-line in-tube SPME coupled to pCEC or mu HPLC. The evaluation of this hyphenation was first carried out for in-tube SPME-mu HPLC with analytical capillaries packed with 3 pm octadecyl silica (ODS). Theobromine (TB), theophylline (TP), and caffeine (CA) were chosen as model drugs for an easy comparison with the results obtained by in tube SPME-mu HPLC. The detection limits of these three analytes were improved more than 100 times when compared with the direct analysis by mu HPLC. Then in-tube SPME-pCEC with CEC capillaries packed with perphenylcarbamoylated P-CD-bonded silica particles was applied to the determination and analysis of propranolol enantiomers in human urine. Under optimal extraction and separation conditions, the experimental LODs were 4 and 7 ng/mL for (S)-propranolol and (R)-propranolol, respectively. The calibration curves showed good linearity for both (S)-propranolol (R-2 = 0.9997) and (R)propranolol (R-2 = 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.