Superoxide anion (O₂^.-), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O₂^.- level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O₂^.- assay by monitoring the unique product 2-OH-E⁺ of HE/O₂^.- reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O₂^.- formation. By the detection of 2-OH-E⁺, we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O₂^.-. Firstly, we demonstrated the proportionality of HE/O₂^.- reaction. Secondly, ungerimine was used as internal standard to eliminate daily basis and matrix effect in the LC/MS-based detection of 2-OH-E⁺. Afterward, the total protein concentration was utilized for cell number normalization. Accordingly, an equation was further proposed to calculate the relative abundance (RA) of intracellular O₂^.-. Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O₂^.- generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O₂^.- assay method is more sensitive, selective and accurate.