The formation of thio-succinimide hydrolysis isomeric products is well known, however, the differentiation between them has not been sufficiently studied so far. This work demonstrated a reference material-free characterization of hydrolyzed thio-succinimide isomers using EAD. Similar to the differentiation between aspartic acid and iso-aspartic acid generated from asparagine deamidation, we observed distinctive [R1+Thio+57+H] + , [R2+Succ+H2O-57+H] + for thio-isoAsp and enhanced [R2+Succ+H2O-44+2H] 2+ for thio-Asp in EAD MS/MS spectra. However, [M-44+H] + and [M-60+H] + failed to serve as diagnostic ions due to their similar intensities in EAD MS/MS spectra for both thio-Asp and thio-isoAsp. To our surprise, we observed a significantly enhanced CO2 neutral loss after formation of the [R2+ThioSucc+H2O+2H] 2+ product ion for thio-isoAsp. This product ion is denoted as [R2+ThioSucc+H2O-44+2H] 2+ , and for the very first time, we report it as a unique diagnostic ion for differentiating thio-Asp and thio-isoAsp. To our knowledge, a similar product ion has not been identified for asparagine deamidation. The formation of this product ion might relate to the thiol group linked to succinimide ring and the structures of R2. For instance, in the case of deamidation, the succinimide ring is linked with amino acids at both ends. Compared to the conventional approach which matches RT of purified reference material to distinguish isomers, EAD serves as a novel tool to identify isomeric structures without reference standards. The differentiation between thio-Asp and thio-isoAsp may eventually benefit the drug development where thiol-maleimide conjugation is employed.