Poly(ADP-ribose) polymerases (PARPs) play important roles in various biological processes, including DNA repair, transcriptional regulation, mitosis, and RNA processing. PARP inhibitors are in clinical trials for treating human cancers. Understanding the biological function of PARPs will be important to fully realize the therapeutic potential of PARP inhibitors. We have developed a clickable analogue of nicotinamide adenine dinucleotide (NAD) that can be used for in-gel visualization, affinity purification and identification of substrate proteins of PARPs. The protocols in this article describe a general procedure to label substrate proteins of PARPs using the clickable NAD analogue.