Posttranslational modification of proteins (PTMs) offers a versatile mechanism to fine-tune the structure, activity, and interactions of the target proteins. Understanding the dynamics and prevalence of the PTM at the site-specific level will provide mechanistic insight into the physiological significance of the modification pathway in cells and diseases. In this chapter, we describe a highly efficient chemical proteomic workflow for the absolute quantification of lysine acetylation stoichiometry. The strategy is capable of measuring the site-specific prevalence of acetylation in a system-wide and untargeted manner. We highlight the importance of validating the workflow using standard proteins and synthetic peptides. Detailed protocols for global stoichiometric analysis of lysine acetylation from cell lysate are presented.