The Nielsen group develops new methodologies enabling proteome-wide characterizing of underexplored post-translational modifications. Recently the group established a methodology that allows unbiased studies of endogenous ADP-ribosylation in a systems-wide manner. Here we describe usage of the Af1521 methodology for characterization of the ADP-ribosylation regulome as a function of cellular regulation based upon stimulus, time, and enzymatic perturbations. To this end, we identified and quantified thousands of ADP-ribosylation sites and determined their temporal dynamics after stimulating human cells with various types of genotoxic stresses, and profiled the regulation of the ADP-ribosylome upon cellular deletion of HPF1 and ARH3. We furthermore demonstrate the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for improved detection and quantification of ADP-ribosylation, hereby advancing the study of ADP-ribosylation under physiological conditions and opening up new analytical directions for the integrative analysis of the ADP-ribosylation regulome. Collectively, we present a proteomics-based characterization of the ADP-ribosylation regulome and provide new insights into the global, integrative view of cellular regulation of this important protein modification.