Malonyl-CoA is a biochemically important compound, formed by an acetyl-coenzyme A carboxylase catalysed reaction. The stability of this short-chain coenzyme A derivative under various experimental conditions is discussed in this article. High-performance liquid chromatography was used for the analysis of the reaction mixture because of its excellent selectivity and sufficient sensitivity. Several variables were investigated as possible stability-influencing factors: pH, magnesium and buffer concentration, reaction temperature and time. The Plackett-Burman screening design was first used for selecting the most important variables, with which a central composite design was constructed. In this way, a response surface was obtained with the percentage remaining malonyl-CoA as a function of magnesium concentration, reaction temperature and time. The usefulness of this approach is demonstrated by obtaining kinetic data from the mathematical function and by the evaluation of the stopping of reaction procedure in the activity assay of acetyl-coenzyme A carboxylase.