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Rationale: Deuterium‐labeled organic compounds, reagents, and drugs are widely used in many scientific research fields. Isotopic purity as the feature parameter of deuterated compounds is of great importance. In this article, we used electrospray ionization with high‐resolution mass spectrometry (ESI‐HRMS) to study isotopic purity of deuterium‐labeled organic compounds based on assigning and distinguishing the corresponding H/D (hydrogen–deuterium) isotopolog ions of deuterated compounds. Methods: We systematically considered the specific requirements of accuracy and resolution of ESI‐HRMS when measuring isotopic purity and demonstrated some actual cases using ESI‐HRMS and ultraperformance liquid chromatography (UPLC)‐HRMS. Meanwhile, ESI‐HRMS/MS of deuterated compounds was performed to obtain more information on deuterium‐labeled position characterization. Results: Two isotopic purity calculation methods based on the relative abundance in ESI‐HRMS and UPLC‐HRMS of H/D isotopolog ions (D0–Dn) were compared, which gave consistent isotopic purity values and were in good agreement with the certified isotopic purity values. We further studied and monitored the H/D exchange reaction of ethyl 3‐(4‐bromophenyl)‐3‐oxopropanoate (EBPO) by evaluating the dynamic isotopic purity changes in EBPO‐D2 in the H/D exchange reaction in situ. Conclusion: The isotopic purity characterization methods using ESI‐HRMS discussed in this article have some outstanding advantages: rapid, highly sensitive, very low sample consumption (even below nanogram level), and deuterated solvent‐free. Thus, this low‐impact analytical method requires less time and is cost effective and might have good application potential for in‐situ isotopic purity analysis and for monitoring the H/D exchange reactions. [ABSTRACT FROM AUTHOR]