Multi-site phosphorylation is an important mechanism for achieving intricate regulation of protein function. Here, we have extended the AQUA methodology (Absolute Quantification of Abundance) and validated its applicability to quantitatively study multi-site phosphorylation. As a test case, we chose the conserved inhibitory site of the Cyclin Dependent Kinases (CDKs), Cdk1, Cdk2 and Cdk3, which are important regulators of cell-cycle transitions and apoptosis. Inhibitory phosphorylation at threonine-14 (T14) and tyrosine-15 (Y15) of the CDKs is modulated by complex regulatory mechanisms involving multiple kinases and phosphatases. Yet, the resulting quantitative dynamics among the four possible phosphorylated and non-phosphorylated versions of CDKs (T14p-Y15p, T14p-Y15, T14-Y15p, and T14-Y15) has not been investigated to date. Hence, we used the heavy isotope labeled tryptic peptides spanning the inhibitory site as internal standards and quantify all the four versions by LC-SRM (Liquid Chromatography-Selected Reaction Monitoring). Quantification of the phosphorylation status of the inhibitory site in the cell extracts provided novel quantitative insights: 1) the transition to mitotic phase is dominated by the conversion of ‘T14p-Y15p’ to the ‘T14-Y15’ form whereas the two mono-phosphorylated forms were considerably lower in abundance. 2) Amount of all four forms decreased during the progression of apoptosis, but with differing kinetics. Analysis of immunoprecipitated Cdk1 and Cdk2 revealed that the inhibitory site phosphorylation state of both kinases at different stages of the cell cycle follows the same trend. Quantitative immunoblotting using antibodies to Cdk1 and Cdk2, and to the T14-Y15p form suggested that quantification by AQUA was reliable and accurate. These results highlight the utility of internal standard peptides to achieve accurate quantification of multi-site phosphorylation status.