A rapid and sensitive LC–MS/MS method has been developed for quantitative analysis of cardiolipin (18:2)4 or CL (18:2)4 in human leukocytes and mouse mitochondria. The structural analog CL (14:0)4 was used as the internal standard. Both CL (18:2)4 and the IS were extracted using a modified Folch method, and separated on a Waters XBridge® BEH C18 XP column using a mobile phase of 0.1% ammonium hydroxide in acetonitrile/water (90:10, v/v) pumped at a flow rate of 0.4mL/min. Quantitation was achieved by negative ESI-MS/MS in MRM mode. The total run time was 2.00min with retention times of 0.74min for the IS and 0.84min for CL (18:2)4, respectively. The method was validated according to the US-FDA guidance for bioanalytical method validation using human leukemia and lymphoma cell lines, which had a calibration range of 0.120–60.2nM with a correlation coefficient >0.999. The intra- and inter-assay accuracy and precision were ≤±5% and ≤8%. The IS normalized matrix factors of CL (18:2)4 and the IS normalized recoveries of CL (18:2)4 ranged 0.92–1.04, and 95–101%, respectively. The stability studies showed that CL (18:2)4 was stable under various test conditions. The developed method was successfully applied to the measurement of CL (18:2)4 in various biological samples including K562 and HL-60 human leukemia cell lines, U937 human lymphoma cell line, white blood cells from patients of Alzheimer’s disease and normal cognitive controls (NCCs), and mitochondria from mouse skeletal muscles. It may be useful for preclinical and clinical studies of this compound.