The protein X-linked inhibitor of apoptosis (XIAP) plays an important role in caspase inactivation and as a consequence in the inhibition of apoptosis. It is known that IAP antagonists are able to specifically bind XIAP and reactivate caspase activity stimulating apoptosis. The viral IAP-associated factor (VIAF) protein is a novel IAP-interacting factor able to modulate caspase activation during apoptosis. We show that the C-terminal domain of VIAF (c-VIAF) is not able in vitro to behave as a direct IAP antagonist. By [15N-1H] HSQC NMR studies we revealed that c-VIAF binds to the RING domain of XIAP and characterized the important residues involved in the binding. Through 1D 1H-NMR screening of 1000 compounds from an in-house collection, we found that compound BI-86-E10 is able to bind to c-VIAF in a region adjacent to that interacting with the RING domain of XIAP, as supported by homology modeling and computational docking studies. After an initial round of SAR, the compound analog BI-86-E6 was found to bind with a K(d) value of 16.5 microM. These initial compounds may serve as chemical probes for further functional studies, possibly aimed at validating c-VIAF as a novel target for antiviral drug development.