ADP-ribosylation is a dynamic post-translational modification predominantly catalyzed by a large family of enzymes called PARPs. PARPs are implicated in diseases such as cancer and inflammation, however, the role of ADP-ribosylation in these contexts is unclear, in large part due to the lack of appropriately selective inhibitors. In this dissertation, I describe a new strategy to develop potent and selective inhibitors for PARPs, by exploiting non-conserved cysteine residues. I also describe a new dual post-translational modification between ADP-ribosyaltion and ubiquitylation, called mono-ADP-ribosyl ubiquitylation, or MARUbylation, which is induced by innate immune responses. The findings presented in this dissertation open up many new directions for the PARP field—both in terms of inhibitor development and post-translational regulation.