Abstract The most common adult brain tumor is Glioblastoma Multiforme (GBM), an extremely aggressive cancer with only scant treatment options. Even with standard of care most patients present with a recurrence and the median survival is only circa 15 months. The need, therefore, for new therapeutic targets and treatment options is pressing. Here we describe here a multipronged approach to identifying said targets. We present an established methodology for the isolation and culture of patient derived GBM samples that retain the “stem-like” fraction thought to underlie resistance and recurrence. Furthermore we show genomically that these samples represent specific subtypes of the disease yet still form distinct groups in unbiased clustering analysis. Thus we have multiple representative patient derived cultures that are suitable for our drug discovery and chemical biology analyses. Using a process we term Chemical Biology Fingerprinting (CBF) we utilize small focused, and clinically relevant, chemical collections in order to identify patterns of chemovulnerabilities across multiple samples. This allows an unbiased yet cancer relevant sub-stratification and the identification of agents, and therefore targets, which may be relevant for GBM patient subtypes. Indeed our use of the highly annotated NCI CTD2 Informer Set of chemicals allows ready drug-to-target mapping and facilitates data sharing across the CTD2 network. Moreover, already defined subgroups can be clustered to find agents, or groups of agents, that show selective activity against traditional classifications (e.g. proneural, mesenchymal etc.). Finally our strategy is permissive for the identification of “exceptional responders”. That is, individual patient samples that respond to a specific drug whilst most samples are refractory. In sum we demonstrate generation of patient derived models and identify specific, and novel, drugs that may be relevant for specific GBM subtypes. Supported by NIH U01CA168397 Citation Format: Darren Finlay, Pedro Aza-Blanc, Harshil Dhruv, Alexey Eroshkin, Craig Hauser, Jeff Kiefer, Seungchan Kim, Tao Long, Robert G. Oshima, Sen Peng, Gil Speyer, Michael Berens, Kristiina Vuori. Novel target discovery for glioblastoma using chemical biology fingerprinting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1142. doi:10.1158/1538-7445.AM2017-1142