As one of the most important post-translational modifications (PTMs), protein adenosine diphosphate ribosylation (ADP-ribosylation) serves as key effectors and regulators in a plethora of biological processes. Although the biological roles of mono-ADP-ribosylation (MARylation) and poly-ADP-ribosylation (PARylation) have been extensively studied, and many relevant inhibitors have been developed or marketed, drug development for two classes of ADP-ribose-binding proteins has been lacking. The first one is viral macrodomains (Mac), which can bind and hydrolyze host cell protein MARylation induced by interferon (IFN) signaling and thus counter host immune response. Although several small-molecule inhibitors have been reported for some viral macrodomains, their affinities are all in the micromolar range and none of them possess both high activity and satisfactory drug-like properties. The other overlooked ADP-ribose-binding domain is the PAR-binding WWE domains present in multiple E3 ligases that have important cancer implications, but no WWE domain inhibitor or relevant screening method has been reported. Therefore, for both macrodomains and WWE domains, probes and inhibitors are highly desired to elucidate their function and druggability. In Chapter 2, I developed a robust fluorescent polarization (FP) assay that works for a variety of human and viral macrodomains, using an ADPr-based tracer molecule. In Chapter 3, I further optimized the tracer by incorporating the structure of GS-441524, an adenosine mimic known to bind SARS-CoV-2 Mac1. The optimized probes show hundreds to thousands-fold affinity increase for viral macrodomains, enabling cost-effective high-throughput screens. In Chapter 4, I constructed a focused library of GS-441524 derivatives in 96-well plates leveraging the simple and high-yielding amide-coupling reaction. The crude reaction mixtures were directly screened in the FP assay developed in Chapter 3. Several hits were identified and further structurally optimized to nanomolar inhibitors for both SARS-CoV-2 and MERS-CoV macrodomains with favorable drug-like properties. In Chapter 5, I designed and synthesized an isoADPr-based FP probe with high binding affinity for the WWE domain of RNF146, a key activator of the oncogenic Wnt signaling pathway. A facile inhibitor screening method was developed with this FP probe, which will be useful for the future screening of RNF146 WWE domain inhibitors.