Helicases, described as 'guardians of the genome', are enzymes that unwind DNA or RNA. Certain helicases, such as Bloom syndrome protein (BLM), are highly implicated in the DNA damage response (DDR). For example, BLM has various roles in homologous recombination (HR), a key pathway in the repair of DNA double strand breaks. Targeting proteins involved in DDR has recently garnered potential as a selective chemotherapeutic strategy. In this work, a tool inhibitor targeting BLM helicase was sought to increase understanding of its role and relevance in DNA damage repair. The only characterised BLM helicase inhibitor, ML216, was developed from the results of a quantitative high-throughput screen (qHTS) screen on BLM helicase.1 This reported inhibitor presented as a good candidate for a starting point in a hit optimisation campaign that ultimately was not successful due to poor physicochemical properties of this compound series. In order to identify a more suitable hit optimisation candidate, 672 actives from the MLSMR qHTS screen [AID2528]2 were analysed and 7 series were selected for in-house synthesis and biological evaluation. This led to the discovery of a single series that is a relatively potent inhibitor of BLM helicase. Hit optimisation primarily focused on the design and synthesis of a highly potent compound with a good solubility profile. Subsequently, co-crystallisation of BLM with this novel series led to the application of rational structure-based methods in the design of tool BLM inhibitors.