Key-players in the intrinsic apoptosis pathway are the B-cell lymphoma protein 2 (Bcl-2) family of proteins. Despite the opposing pro- or anti-apoptotic functions of the various family members, they all exhibit a high degree of sequence and structural similarity in their “Bcl-2 homology regions”, in particular, the presence of a highly conserved hydrophobic ligand binding groove. Both pro-apoptotic and anti-apoptotic proteins within the Bcl-2 family have long been considered targets for new anti-cancer drug development. Therefore, characterising interactions between the proteins and hit/lead compounds identified by fragment-based or high throughput compound screens at the level of individual amino acid residues is a critical step for lead/ligand optimization, and is at the heart of structure-based drug discovery. Here, we report the backbone chemical shift assignments for one of the anti-apoptotic proteins, Bfl-1, and NMR titration results for the validation and quantification of several low-molecular weight hits arising from fragment-based or high throughput compound screens. Compound binding to Bfl-1 was observed proximal to its hydrophobic groove whilst titration experiments enabled confirmation of binding affinity.