Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-DM1). A convenient liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 μm) with mobile-phase gradient elution. The calibration curves of each analyte ranging from 1 to 100 nM showed good linearity (r2 > 0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T-DM1.