Abstract: Pidotimod (3-L-pyroglutamyl-L-thiaziolidine-4 carboxylic acid) is a synthetic dipeptide molecule with immunomodulatory properties. Since the early 90’s, a wide scientific production has been published about it. As it’s effective in the treatment of people infected with bacteria and viruses, it is used in the treatment of repeated infections of the respiratory, urogenital, and ear, nose, throat systems. Pidotimod therapy is a reliable, simple, and safe approach to treat children with recurrent respiratory infections. Liquid chromatography-mass spectrometry (LC/MS) method has been used for determination of pidotimod in different biological matrices. Only a few HPLC-UV methods have been reported for the quantification of the related substances in pidotimod and its formulation. The reported LC/MS methods usually use formic acid or acetic acid in the mobile phase which are not suitable for UV detector, and the separation is not feasible for the related substances. And the reported HPLC-UV methods usually use phosphate and phosphoric acid in the mobile phase which are not suitable for mass spectrometry detector. However, there is no reliable LC/MS method for identification of the related substances in pidotimod. In this paper, a LC/MS method was developed for the separation and characterization of process related substances and the major degradation products in pidotimod. Electrospray positive ionization high resolution time-of-flight mass spectrometer (ESI-TOF MS) was used for the determination of the accurate mass and elemental composition of the parent ions of the related substances, and triple quadrupole tandem mass was employed for the product mass spectra determination. The separation was performed on a Sepax GP-C8 column (250 mm×4.6 mm×5 μm) using 0.005% trifluoroacetic acid (TFA) in water as mobile phase A and methanol as mobile phase B. The flow rate was 1.0 mL/min and the column temperature was set at 30 ℃. Analytes were monitored at 210 nm. The injection volume was 20 μL. Pidotimod was tending to degrade under acid, alkaline, oxidative, thermal stress and photolytic stress. The structures of the related substances were then figured out through elucidation of the fragment ions. A total of 11 related substances were detected and characterized, including process related substances (related substances 1 and 4) and degradation products. Related substances 4 and 10 were identified by synthetic reference and related substances 1, 2, 8 and 9 were reported before this study, while related substances 3, 5, 6, 7 and 11 were reported for the first time. The method is effective for separation and identification of the related substances of pidotimod, and the results are useful for the quality control and process optimization of pidotimod.