Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MSbased techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [␥- 18 O 4 ]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min ؊1 to 0.001 min ؊1 . Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680. Molecular & Cellular Proteomics 13: 10.1074/mcp.O113.036145, 1106-1118, 2014.Protein phosphorylation is crucial for modulating protein structure, protein localization, and the protein-protein interactions that form the basis of many cell-signaling networks. Phosphorylation-based signaling often takes the form of a cascade in which sequential protein phosphorylations lead to changes in protein stability, function, and localization. Protein kinases, the enzymes that propagate these signals, catalyze the transfer of ␥ phosphate from ATP onto serine, threonine, or tyrosine residues of substrate proteins. The sites of protein phosphorylation and phosphorylation dynamics are important in determining the biological outcome of a signaling event (1). For instance, protein phosphorylation drives many of the changes during the cell cycle (2, 3). During mitosis, kinases are activated at precise times to direct the course of chromosome segregation and cell division. For example, CDK1 activation at the beginning of mitosis leads to phosphorylation of NUP98 during prophase, w...