A novel HPLC method for the determination of the impurities in desonide cream was established and validated for the further improvement of the official monograph in USP. Desonide was well resolved from the photodegradation impurity, which overlapped with desonide in USP method. The method was validated in accordance to the regulatory guidelines recommended by the International Conference on Harmonisation and this validation included specificity, limit of detection, limit of quantification, linearity and accuracy. Four degradation impurities in desonide cream were characterized by a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) in positive mode of electrospray ionization. Through the multiple heart-cutting 2D LC approach and online demineralization technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely, and the TIC chromatogram of LC–MS could be in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities. In the first dimension, the column was Phenomenex Kinetex C8 (4.6 mm × 150 mm, 2.6 μm) with a non-volatile salt mobile phase. In the second dimension, the column was Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 μm) with a volatile salt mobile phase. The structures of four degradation impurities in desonide cream were deduced based on the HPLC-MSn data. The established method in this study was simple and reliable for routine quality control of desonide cream.