Cardiolipin is an important cellular component, and its normal level is a key prerequisite for maintaining the structure and function of mitochondria. To accurately quantify endogenous cardiolipin content in mitochondria, a standard addition method (SAM) was developed to establish a high-performance liquid chromatographic (HPLC) technique that is both reliable and accurate. Increasing amounts of cardiolipin standards were added to a constant amount of isolated mitochondria prior to the extraction procedure, and the two were extracted together. By limiting the interference effects that occur to within an acceptable range in an analytical system examined, this procedure ensures an ideal match of the sample composition in the standards, even if the composition is extremely complex or completely unknown. Then, the desired results can be obtained by extrapolation. As such, the authentic content of the endogenous cardiolipin can be obtained with greater accuracy than with classical detection methods, e.g. external standard calibration (ESC) and internal standard calibration (ISC). This method provides an excellent means of quantifying endogenous substances in living cells. The authors expect this method to be useful for researchers working on mitochondria-related mechanisms, cell survival-related mechanisms and similar topics.