Covalently modifying a protein has proven to be a powerful mechanism of functional regulation. N-epsilon acetylation of lysine residues was initially discovered on histones and has been studied extensively in the context of chromatin and DNA metabolism, such as transcription, replication and repair. However, recent research shows that acetylation is more widespread than initially thought and that it regulates various nuclear as well as cytoplasmic and mitochondrial processes. In this review, we present the multitude of non-histone proteins targeted by lysine acetyltransferases of the large and conserved MYST family, and known functional consequences of this acetylation. Substrates of MYST enzymes include factors involved in transcription, heterochromatin formation and cell cycle, DNA repair proteins, gluconeogenesis enzymes and finally subunits of MYST protein complexes themselves. Discovering novel substrates of MYST proteins is pivotal for the understanding of the diverse functions of these essential acetyltransferases in nuclear processes, signaling, stress response and metabolism.