The phosphoinositide 3-kinase (PI3K)/Akt pathway controls a vast array of normal physiological processes and is frequently aberrantly activated in cancer, thus identifying PI3K/Akt-signaling components as promising drug targets in oncology. However, implementation of rational cancer therapies for this pathway needs robust and simple tools to stratify patients according to PI3K pathway activation and to validate and measure the impact of targeted inhibition on primary cancer tissues. Herein we present a technique for the quantification of the PI3K/Akt-signaling pathway based on the mass spectrometric measurement of PI3K-dependent protein kinase activity in cell lysates. The concept of this application of MS is to exploit enzymatic activity to amplify the signal of the enzyme under study analogous to the PCR used to amplify nucleic acid sequences. We show that this approach allows quantitative analysis of a cell-signaling pathway with high sensitivity, precision of quantification, and specificity. Due to its special analytical capabilities and potential for multiplexing, this approach could contribute significantly to cell-signaling studies and to the development and implementation of personalized cancer therapies.