Human RAD52 (hsRAD52) plays a pertinent role in Homology Directed Damage Repair (HDDR) and in supporting faithful genome duplication, but there is still high ambiguity in what biochemical activities and cellular function underlie this role. There is much known about Rad52 activities in yeast cells (saccharomyces cervisiae, scRad52), which functions by annealing of two complementary single stranded DNA (ssDNA) molecules bound by Replication Protein A (RPA), and also serves as a recombination mediator which replaces scRPA with scRad51 recombinase. ScRad52 uses these two biochemical activities to maintain genomic integrity after double stranded breaks via two mechanisms, homologous recombination (HR) and single stranded annealing (SSA). There are similarities between scRad52 and hsRAD52, but the two proteins are no identical. Biochemically, hsRAD52 has shown to mediate annealing of hsRPA-coated ssDNA, but demonstrated no detectable recombination mediator activity, the role played in human cells by BRCA2 tumor suppressor protein. In healthy mammalian cells RAD52 is expendable, but its inactivation leads to cell death when combined with deficiencies in the tumor suppressor genes BRCA1 and BRCA2 commonly mutated in hereditary and sporadic breast cancers. This phenomenon is known as synthetic lethality; which is the simultaneous inactivation/mutation of two or more genes that lead to cell death, whereas the inactivation/mutation of only one of these genes does not affect the cells viability. Synthetic lethality between hsRAD52 deletion and BRCA-deficiency makes RAD52 an attractive breast cancer drug target, and specifically RAD52 ability to bind ssDNA in a geometrically distinct wrapped configuration, which may be important for all of its critical cellular functions. Using Forster resonance energy transfer, FRET-based high throughput screening (HTS) assay we have several small-molecule compounds that perturb the RAD52-ssDNA complex. Here, I will describe detailed biochemical characterization of several of the identified compounds and their ability to perturb hsRAD52-ssDNA complex. The obtained knowledge (structures of the compounds combined with their potency) will be used in the ligand centered cheminformatics approaches to identify novel potent inhibitor scaffolds. These scaffolds will be further developed into anti-cancer drug and molecular probes for dissecting hsRAD52 cellular role.