Protein modifications are biologically important events that may be studied by mass spectrometry-based high-throughput proteome analyses. In recent years, several new technologies have emerged that have widened and deepened the targeted analysis of one important, albeit functionally ill-defined modification, namely protein acetylation. This modification can take place both co- and post-translationally by the transfer of acetyl groups under the catalysis of acetyltransferases. The acetyl group can modify either the α-amino group at the N-terminus, so-called N-terminal acetylation, or the ε-amino group on the side chain of lysine residues. Here, we review several emerging targeted technologies to chart both N-terminal acetylation as well as acetylation at the lysine side chain, on a proteome-wide scale, highlighting in particular studies that have expanded the biological knowledge on the appearance and function of these common but functionally still less investigated co- and post-translational modifications.