In response to DNA damage, cells have evolved mechanisms to halt cell cycle progression, activate repair, or initiate apoptosis. These DNA damage response (DDR) pathways are critical for cellular survival in response to genomic insult, and play important roles in growth, development, and disease. Historically, mediators of DNA damage response signaling have been studied one or a few proteins at a time. Advances in mass spectrometry instrumentation and enrichment methods now allow for more global analysis of the DDR in cells and tissues. In this review we will discuss current methods in liquid chromatography tandem mass spectrometry (LC-MS/MS), enrichment strategies, and targeted analyses for the study of damage signaling. These methods have allowed a greater understanding of the DNA damage response and have highlighted the far-reaching effects of activation of damage-induced pathways.