Cardiac troponin I (cTnI) is the biomarker of choice and considered a gold standard for the diagnosis of acute myocardial infarction. However, the quantitative results of cTnI assay kits from different manufacturers are not comparable. Based on the H/D exchange mass spectrometry (HDX-MS) workflow, we developed an in-vitro diagnostic reagent antibody evaluation strategy to analyze the interactions of epitopes and antibody cocktails─(R195, F12, S13) and (D1, D2, pAb2). The HDX results indicate that the quantitative result bias of the different reagents originates from the ability of antibodies to recognize various cTnI complex forms, such as free cTnI, hydrolyzed cTnI, and cTnI combined with cTnT or TnC as binary or ternary complexes (cTnIC, cTnTIC), in blood based on different epitopes. The data obtained from the peptide HDX of interest after treatment with various antibody cocktails clearly indicated epitope specificity. The consistency of quantitative results can be improved by a thorough investigation into the epitopes recognized by the antibodies of various diagnostic kits, which will lead to the standardization of cTnI diagnosis.