The quality control of cationic cell-penetrating peptides was performed.

Evaluated chromatographic systems differed in particle size, acidic modifier and temperature.

Obtained peptide impurity profile is dependent on the applied chromatographic system.

UHPLC–UV method with TFA as acidic modifier and run at 30 °C was optimal method.

Single quad mass spectrometry detectors offer benefits in the quality control of these peptides.