A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was developed for the determination of acetyl coenzyme A in grape berries using n-propionyl coenzyme A as an internal standard (IS). The sample was extracted with water and then centrifuged for 5 min at 10 000 r/min on a centrifuge at 4 degrees C, and cleaned-up with a C18 solid phase extraction cartridge. The identification and quantification were carried out by using electrospray ionization in positive ion mode (ESI +) with multiple reaction monitoring (MRM). The total run time was 1 min and the elution of both acetyl coenzyme A and n-propionyl coenzyme A occurred at about 0.45 min. This was achieved with a mobile phase consisting of 5 mmol/L ammonium formate-acetonitrile (20: 80, v/v) at a flow rate of 0.4 mL/min on a C18 column. A linear response function was established for the concentration range of 1 - 2 000 microg/L for acetyl coenzyme A and the correlation coefficient was more than 0.99. The limit of detection of acetyl coenzyme A was 0.1 microg/L. The spiked recoveries at 50, 500, 1 000 microg/L ranged from 82.87% - 89.67% with the relative standard deviations less than 10%. This method is simple, rapid, sensitive and can significantly reduce the loss of acetyl coenzyme A during the experiment. It is suitable for the determination of acetyl coenzyme A in grape berries.