ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5′-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.