A simple, sensitive, specific, and cost-effective analytical methodology was developed for the analysis of human plasma samples spiked with imatinib by CZE with on-line UV detection in the context of Therapeutic Drug Monitoring. Several analytical conditions such as the ionic strength (I) and the pH of the BGE composed of citric acid and epsilon-amino caproic acid were studied in regards of the presence of sodium chloride (NaCl) in plasma samples (1% m/v). Computer simulations (Simul software) were used to confirm the experimental results and to understand imatinib electrophoretic behavior in the presence of NaCl. Furthermore, the advantages of adding ACN to the sample containing NaCl to combine efficient protein precipitation and on-line CZE stacking of imatinib were demonstrated. LOD and LOQ values of 48 and 191 ng/mL were obtained from plasma sample supernatant after protein precipitation with ACN, which is much lower than mean imatinib plasma level observed for patients treated by imatinib mesylate (about 1000 ng/mL). Good linearity was obtained in the concentration range 191-5000 ng/mL (R-2 > 0.997). RSD of less than 1.68% and 2.60% (n = 6) for migration times and corrected peak areas, respectively, were observed at the LOQ.