Tetracosactide is a synthetic peptide analogue of the human adrenocorticotropic hormone that stimulates the production of cortisol in the adrenal cortex. The medical use of the compound is primarily the diagnosis of the adrenal cortex function. In order to characterize impurities of the drug, tetracosactide samples were analysed by both liquid chromatography and capillary electrophoresis coupled to a quadrupole time-of-flight mass spectrometer. The identification of the impurities was carried out based on accurate mass determination and fragment ion spectra. The presence of several peptides of lower and higher masses than tetracosactide could be shown, including N- and C-terminally truncated peptides as well as peptides which still contained protecting groups or additional amino acids. Furthermore, a semi-quantitative estimation of the relative amounts of the impurities in different samples as well as a commercial preparation revealed that the number and the type of the impurities varied between the samples. Comparing the selectivity of liquid chromatography and capillary electrophoresis regarding the separation of tetracosactide impurities, it can be stated that capillary electrophoresis showed a higher suitability for the separation of tetracosactide fragments (smaller peptides) while the larger peptides, i.e. those wearing protecting groups, were separated more efficiently by liquid chromatography.