trans-Ketoconazole was identified and quantified as impurity of cis-ketoconazole, an antifungal compound, by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS). The chirality of this impurity was demonstrated separating their enantiomers by adding heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin to the separation buffer in capillary electrophoresis (CE) with UV detection. However, MS detection was hyphenated to the CE instrument for its identification. As both compounds are diastereomers, they have the same m/z values and are needed to be separated prior to the MS identification. A 0.4M ammonium formate separation buffer at pH 3.0 enabled the separation of the impurity from cis-ketoconazole. Under these conditions, the optimization of ESI-MS parameters (composition and flow of the sheath-liquid, drying temperature, drying gas flow, and capillary potential) was carried out to obtain the best MS sensitivity. CZE-ESI-MS optimized conditions enabled the identification of trans-ketoconazole as impurity of cis-ketoconazole. In addition, the quantitation of this impurity was achieved in different samples: cis-ketoconazole standard and three different pharmaceutical formulations (two tablets and one syrup) containing this standard. In all cases, percentages higher than 2.0 were determined for the impurity. According to ICH guidelines, these values required the identification and quantitation of any impurity in drug substances and products.