Protein phosphorylation plays a critical role in a variety of cellular functions. As a result, the monitoring of phosphoproteins in cells represents an important goal for proteomics research. To facilitate phosphoprotein detection, the first enzymatic phosphorylation-dependent biotinylation reaction of proteins is described. Specifically, kinase enzymes were coupled with an ATP-biotin conjugate to efficiently biotinylate substrate peptides and proteins after phosphate transfer. The kinase-mediated biotinylation reaction enables efficient detection of phosphoproteins in cell lysates or phosphopeptides after trypsin proteolysis, demonstrating its utility for proteomics research. Importantly, the studies reveal the cosubstrate promiscuity of kinase enzymes, laying the foundation for development of new chemical tools targeting the phosphoproteome.