Mono-ADP-ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono-ADP-ribosylated proteins have been described. We report the synthesis of ADP-ribosylated peptides from the proteins histone H2B, RhoA and, HNP-1. An innovative procedure was applied that makes use of pre-phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP-ribosylated peptides, thus showing that the sequence surrounding ADP-ribosylated residues affects the substrate selectivity of macrodomains.