This article describes a method for site-specifically incorporating succinylation (SucK) and glutarylation (GluK) into proteins using genetic code expansion. These modifications, which reverse the charge of lysine from +1 to −1, are known to impact protein structure and function but have been challenging to study due to the difficulty in obtaining homogeneously modified proteins. The authors synthesized thioester derivatives of SucK and GluK that mask the negatively charged side chain carboxylate, allowing for their site-specific incorporation into proteins using an evolved pyrrolysyl-transfer RNA synthetase (PyRS) variant. This method was successfully applied to various bacterial and mammalian target proteins, including non-refoldable multidomain proteins. The study demonstrates that these modifications can modulate the enzymatic activity of metabolic enzymes, regulate protein-protein interactions in ubiquitin signaling, and influence DNA clamp loading and replication. The approach provides a robust tool for understanding the functional roles of these post-translational modifications in biological processes.This article describes a method for site-specifically incorporating succinylation (SucK) and glutarylation (GluK) into proteins using genetic code expansion. These modifications, which reverse the charge of lysine from +1 to −1, are known to impact protein structure and function but have been challenging to study due to the difficulty in obtaining homogeneously modified proteins. The authors synthesized thioester derivatives of SucK and GluK that mask the negatively charged side chain carboxylate, allowing for their site-specific incorporation into proteins using an evolved pyrrolysyl-transfer RNA synthetase (PyRS) variant. This method was successfully applied to various bacterial and mammalian target proteins, including non-refoldable multidomain proteins. The study demonstrates that these modifications can modulate the enzymatic activity of metabolic enzymes, regulate protein-protein interactions in ubiquitin signaling, and influence DNA clamp loading and replication. The approach provides a robust tool for understanding the functional roles of these post-translational modifications in biological processes.