An alternative methodology for the determination of ethambutol by capillary zone electrophoresis (CZE) under direct UV detection at 262 nm, using acetic acid/sodium acetate buffer solution (pH 4.6) containing copper(II) sulphate to form the ethambutol-copper(II) complex, within analysis time of 2.5 min is proposed. The optimum CE conditions for the background electrolyte were established performing experiments of a 3(2) factorial design. Complex formation was evidenced by the UV batochromic shift and the [CuETB](0) and [CuETB](2+) chemical structures were indicated by LC-MS analysis. After some validation parameters have been performed, such as linearity (r=0.999), selectivity (comparison between slope of the calibration curve of the external standard and calibration curve of the standard addition), area precision (RSD%: <2.13 for ETB and <1.94 for 2A1B), recovery mean (101.7% for ETB and 99.95% for 2A1B) and quantification limit (mg L(-1): 10.17 for ETB and 19.70 for 2A1B), the method was successfully applied to ETB analysis in pharmaceutical formulation samples. It is possible to determine the presence of the 2A1B impurity at concentrations of less than 1% ETB content.