Capillary electrophoresis (CE) coupled with mass spectrometry (MS) with a sheath liquid interface is nowadays recognized as a powerful separation technique for drugs and metabolites analysis in human urine and can be applied in numerous fields such as clinical toxicology, drug substitution monitoring, forensic sciences and antidoping. With an acidic background electrolyte containing 15 mM ammonium formate at pH 2.5 and a sheath liquid consisting in a mixture of isopropanol/water (50:50, v/v) with 0.5% formic acid, CE-ESI-MS in positive mode demonstrated excellent performance for simultaneous analysis of basic drugs of abuse and metabolites in urine (e.g. cocaine, amphetamine, morphine and phase II metabolites). To achieve the desired level of sensitivity, two injection modes and three sample pre-treatments were evaluated. The detection of basic drugs and phase II metabolites in diluted urine was achieved at concentrations above 1 μg/mL. In order to enhance sensitivity, a sample preparation was required. A liquid-liquid extraction (LLE) was compared with solid-phase extraction. LLE was performed at alkaline pH and samples were electrokinetically injected. A chemometric approach (Doehlert design) was carried out in order to determine optimized injection parameters. Limits of detection (LOD) down to 10 ng/mL were reached with field-amplified sample injection but phase II metabolites were not extracted. Therefore, instead of LLE a SPE was performed on C18 sorbent, and elution fraction after washing step containing phase II metabolites was loaded on mixed-mode anion exchanger cartridges. After electrokinetic injection, this two-step SPE allowed LOD ca. 10 ng/mL for drugs and phase II metabolites.