This study dealt with the potential of MEKC with LIF detection involving derivatization with sulfbindocyanine succinimidyl ester (Cy5) for the separation and determination of P-lactam antibiotics (ampicillin, amoxicillin, cephradine, and cephalexin) in environmental water samples. Water samples of 50 mL were enriched by SPE by passage through a weak basecation Amberlite((R)) IRA-93 exchange column. SDS micelles play important roles in the whole analytical process by improving the yield (sensitivity) and the kinetics of the labeling reaction, the elution of the retained antibiotics from the SPE preconcentration system and the electrophoretic resolution of their Cy5-derivatives. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 10 min and direct injection for MEKC analysis, which is conducted within about 15 min using 15 mM SDS in the running buffer (35 mM sodium borate at pH 9.3). LODs from 30 to 45 ng/L and RSDs (within-day precision) from 3.5 to 5.9% were obtained for the antibiotics in water samples with average recoveries ranging from 96.4 to 99.4%. These results indicate that the method proposed is a straightforward and sensitive tool for the determination of these antibiotics in environmental water samples providing similar quantitative results to those using more expensive equipment like LC-electrospray MS/MS.