Updated project metadata. Posttranslational modifications of proteins (PTMs) greatly enhance their functional diversity, surpassing the number of gene-encoded variations. One intriguing PTM is ADP-ribosylation, which utilizes nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell-permeable NAD+ analogs that are modified with a dye or an affinity tag, respectively, and demonstrate their suitability as tools for investigating of the cellular ADP-ribosylation process. Utilizing desthiobiotin-tagged probes, we characterized ADP-ribosylome changes during oxidative stress in HeLa cells. We identified proteins previously described as ADP-ribosylation targets or closely associated with them. Therefore, we believe that our cell-permeable NAD+ probes offer reliable tools for comprehensive ADP-ribosylation investigations and understanding cellular responses to stress.