Phosphatidylglycerol (PG) and cardiolipin (CL) are synthesized in mitochondria and regulate numerous biological functions. In this study, a novel fluorometric method was developed for measuring PG and CL using combinations of specific enzymes and Amplex Red. This assay quantified the sum of PG and CL (PG + CL) regardless of the species of fatty acyl chain. The calibration curve for PG + CL measurement was linear, and the detection limit was 1 μM (10 pmol in the reaction mixture). This new method was applied to the determinations of PG + CL content in HEK293 cells and CL content in purified mitochondria, because the mitochondrial content of PG is negligible compared with that of CL. We demonstrated that the PG+CL content was greater at low cell density than at high cell density. The overexpression of phosphatidylglycerophosphate synthase 1 (PGS1) increased the cellular contents of PG + CL and phosphatidylcholine (PC), and reduced that of phosphatidic acid. PGS1 overexpression also elevated the mitochondrial contents of CL and PC, but had no effect on the number of mitochondria per cell. In addition to the enzymatic measurements of other phospholipids, this simple, sensitive and high-throughput assay for measuring PG + CL can be used to understand cellular, physiological and pathological processes.