Genomic instability is a critical driver in the process of cancer formation. At the same time, inducing DNA damage by irradiation or genotoxic compounds constitutes a key therapeutic strategy to kill fast-dividing cancer cells. Sensing of DNA lesions initiates a complex set of signalling pathways, collectively known as the DNA damage response (DDR). Deciphering DDR signalling pathways with high-throughput technologies could provide insights into oncogenic transformation, metastasis formation and therapy responses, and could build a basis for better therapeutic interventions in cancer treatment. Mass Spectrometry (MS)-based proteomics emerged as method of choice for global studies of proteins and their posttranslational modifications (PTMs). MS-based studies of the DDR have aided in delineating DNA damage-induced signalling responses. Those studies identified changes in abundance, interactions and modification of proteins in the context of genotoxic stress. Here we review ground-breaking MS-based proteomics studies, which analysed changes in protein abundance, protein-protein and protein-DNA interactions, phosphorylation, acetylation, ubiquitylation, SUMOylation and Poly(ADP-ribose)ylation (PARylation) in the DDR. Finally, we provide an outlook on how proteomics studies of the DDR could aid clinical developments on multiple levels. This article is protected by copyright. All rights reserved.