The dynamic nature of protein posttranslational modification (PTM) allows cells to rapidly respond to changes in their environment, such as nutrition, stress, or signaling. Lysine residues are targets for several types of modifications, including methylation, ubiquitination, and various acylation groups, especially acetylation. Currently, one of the best methods for identification and quantification of protein acetylation is immunoaffinity enrichment in combination with high-resolution mass spectrometry. As we are using a relatively novel and comprehensive mass spectrometric approach, data-independent acquisition (DIA), this protocol provides high-throughput, accurate, and reproducible label-free PTM quantification. Here we describe detailed protocols to process relatively small amounts of mouse liver tissue that integrate isolation of proteins, proteolytic digestion into peptides, immunoaffinity enrichment of acetylated peptides, identification of acetylation sites, and comprehensive quantification of relative abundance changes for thousands of identified lysine acetylation sites.