Poly (ADP‐ribose) polymerases, PARPs, are a family of 17 enzymes (in humans) that were identified based on their ability to transfer ADP‐ribose onto target proteins from nicotinamide adenine dinucleotide (NAD+). The ADP‐ribosylation reaction occurs in a highly conserved PARP catalytic domain found in all fifteen of the active PARPs. PARPs 14 and 15 have recently received increased attention due to their role in viral recognition and innate immunity. Significant effort using modern proteomic methods has led to the identification of thousands of potential PARP targets in the cell. This analysis has not yet uncovered which sites are preferentially targeted in vivo, nor the determinants for PARP family‐member specific targeting. Further, current mass spectrometric analytical methods require proteolysis of the target protein, limiting the study of dynamic ADP‐ribosylation. Herein, we present a matrix‐assisted laser desorption/ionization time of flight (MALDI‐TOF) method using thin‐layer chromatography (TLC) that facilitates population‐wide analysis of ADP‐ribosylation. Using this method, we identify, for the first time, a minimal peptide fragment (18 amino‐acids) that is preferentially modified by PARP14. We have also identified the specific glutamate (E) residue that is targeted on this peptide. Using this method, we also observe divergent ADP‐ribosylation dynamics for the catalytic domains of PARPs 14 and 15, with PARP15 modifying more sites on itself (+3–4 ADP‐ribose) than the closely related PARP14 protein (+1–2 ADP‐ribose) ‐ despite similar numbers of potential modification sites. We further demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARPs 14 and 15 prefer acidic residues. We are utilizing this method – in conjunction with the identified peptide – as a scaffold for the development of first-in-class peptide-based selective PARP inhibitors. The presented findings will be useful in uncovering the basic biochemistry governing ADP‐ribosylation, should find utility in the identification of PARP family‐member specific peptide targets and inhibitors, and will provide new avenues for investigating the role of specific PARP family members during viral infection.