The enzymatic peptide phosphorylation by cAMP-dependent protein kinase A (PKA) was optimized and monitored by means of electrospray ionization mass spectrometry (ESI-MS). The direct detection of phosphorylated peptides by MS renders labeling unnecessary, reduces time and labor, due to less initial sample pretreatment. In this study the phosphorylation of the peptide malantide by PKA was performed in batch and reaction compounds were detected by ESI-MS after the incubation time. The subsequent product quantitation was accomplished by using one-point normalization. Applying this set-up, optimum solvent conditions (such as salt and modifier content), concentrations of essential reaction compounds (such as cAMP, Mg2+ and ATP), and the influence of reaction properties (such as pH and reaction time) were determined. The reaction milieu has to be suitable for both, the enzymatic reaction and the mass spectrometric detection. We found that the modifier content and the pH value had to be changed after the enzymatic reaction occurred. Through the addition of methanol and acetic acid, the reaction stopped immediately and a more sensitive mass spectrometric detection could be obtained simultaneously. Furthermore, an inhibitor study was performed, testing the inhibition potency of three protein kinase A inhibitors (PKIs). IC50 values were determined and used to calculate the Ki values, that were 7.4, 19.0 and 340.0 nmol/L for PKI(6-22)amide, PKI(5-24)amide, and PKI(14-24)amide, respectively. These data vary between factor 4.4 (for PKI(6-22)amide) and 8.3 (for PKI(5-24)amide) compared to the Ki values described in literature. However, the Ki values are in good agreement with the data mainly obtained by fluorescence- or radioactivity-based methods. Nevertheless, our results indicate that ESI-MS is a realistic alternative to radioactivity and fluorescence detection in determining enzymatic activity. Furthermore we were able to illustrate its high potential as a quantitative detection method.