Significance Recent proteomic studies have revealed that lysine acetylation is a global and ubiquitous posttranslational modification. However, in the vast majority of cases the lysine acetyltransferases (KATs) responsible for individual modifications remain unknown. Here we present a unique methodology that connects KATs to their substrates. To validate the methodology, we use the yeast KAT nucleosome acetyltransferase of histone H4 (NuA4) and identify both protein interactions and acetylation targets. Importantly, this methodology can be applied to any KAT and should aid in the linking of KATs to their cellular targets.