[Background] Peptidoglycan (PG) is an important component of the bacterial cell wall. The type VI secretion system (T6SS) can secrete effectors with peptidoglycan hydrolase activities into a neighbor bacterial cell to kill the recipient. However, the enzymatic functions of these effectors have not been fully characterized due to the technical challenges in PG analysis. [Objective] We aim to establish an analytical method using liquid chromatography and mass spectrometry to qualitatively determine the PG-hydrolyzing activities of two Vibrio cholerae effectors TseH and VgrG3. [Methods] The antibacterial effects of TseH and VgrG3 were determined by survival assays and microscopy analysis when ectopically expressed in Escherichia coli. Peptidoglycan was purified from E. coli, and morphologically characterized by transmission electron microscopy (TEM). The ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS) was used to identify the products of peptidoglycan from digestion by TseH or VgrG3. [Results] TEM micrograph showed that the purified peptidoglycan is translucent. Using UPLC-TOFMS to analyze VgrG3-treated PG, we identified three products including disaccharide dipeptide (Di), disaccharide tripeptide (Tri), and disaccharide tetrapeptide (Tetra). [Conclusion] VgrG3, rather than TseH, can digest the β(1-4) covalent bond between N-acetylglucosamine and N-acetylmuramic acid. The established method could facilitate the characterization of other cell-wall targeting antibacterial effectors and chemical compounds.