Since Strahl and Allis proposed the "language of covalent histone modifications", a host of experimental studies have shed light on the different facets of chromatin regulation by epigenetic mechanisms. Initially proposed as a concept for controlling gene transcription, the regulation of deposition and removal of histone post -translational modifications (PTMs), such as acetylation, methylation, and phosphorylation, have been implicated in many chromatin regulation pathways. However, large PTMs such as ubiquitylation challenge research on many levels due to their chemical complexity. In recent years, chemical tools have been developed to generate chromatin in defined ubiquitylation states in vitro . Chemical biology approaches are now used to link specific histone ubiquitylation marks with downstream chromatin regulation events on the molecular level. Here, we want to highlight how chemical biology approaches have empowered the mechanistic study of chromatin ubiquitylation in the context of gene regulation and DNA repair with attention to future challenges. (c) 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).