An automated procedure for the determination of Fructose 2,6-bisphosphate (Fru 2,6-P2) is described. The method based on the degree of activation of the enzyme Pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase from potato tuber by Fru 2,6-P2, uses a discrete-sample automatic analyzer to perform the procedure and to monitor the spectrophotometric changes. The detection limit of Fru 2,6-P2levels is 0.016 pmol (6.25 × 10−14mol ml−1). The coefficients of variation observed when assaying 0.5 nM of Fru 2,6-P2in the reaction mixture, 1.34% and 7.39% (n=10) for the automated and manual method are significantly different. Thus a 5-fold higher precision is shown by the automatic method. The same precision is observed when assaying biological samples from starved or refed animals.