Abstract Chemical proteomics is a rapidly evolving technology that allows for drug target identification for small molecule inhibitors. Here we present an approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules and used to pull-down protein binding partners using the HaloTag technology. CA-modified compounds are cell permeable and have minimal impact on potency, allowing for phenotypic assays of the derivatized compound to be recapitulated. Here we present a study looking at the targets of two compounds - a Brutons Tyrosine Kinase (BTK) small molecule inhibitor and a Phospho-inositol 3 Kinase (PI3K) small molecule inhibitor. BTK is a non-receptor tyrosine kinase expressed in most hematopoietic cells except T cells, it is essential for B cell receptor (BCR) signaling in B cells and as such is the therapeutic focus in many autoimmune diseases, including rheumatoid arthritis (RA) and lupus. Upon binding to growth factor receptor, PI3K is activated which initiates a signaling cascade involving AKT and mTOR that promotes cell cycle progression and cell proliferation. Mutations of the catalytic subunit of PI3KA are observed in several types of cancers. Inhibition of PI3K is therefore an important therapeutic focus. Taselisib, a selective inhibitor of PIK3CA, and G02599124, an inhibitor of BTK, were derivitized with the CA linker. Phenotypic screens compared to the parental compound showed comparable IC50 values. Both CA-compounds showed good binding to HaloTag and were cell permeable in a U2OS stable cell line. Control and test experiments were performed with BTK-CA compound using Jurkat (T cells) and Ramos (B cells) cell lines. Taselib-CA was studied after 1h and 8h treatment in the HCC1954 cell line. Cells were lysed and the CA compounds, together with the bound targets, were rapidly captured onto magnetic resin coated with HaloTag. Unmodified compound was used to competitively elute interacting proteins. Eluted proteins were processed using SDS-PAGE and in-gel digestion. Peptides were analyzed using nanoscale LC-MS/MS coupled with an Orbitrap Velos Pro mass spectrometer. Using this approach we were able to specifically isolate BTK when compared to control experiments in T cells. In the Taselib-CA experiment, we were able to specifically isolate the PIK3CA catalytic subunit and PIK3R1 and PIK3R2 regulatory subunits. Observation of the wild type and mutant forms of PI3KCA will be discussed. Citation Format: Victoria Pham, James Crawford, Steve Saben, Michael Ford, Richard Jones, Danette Daniels, Thomas Kirkland, Marjeta Urh, Jennie Lill. Chemical Proteomic Analysis of BTK and PI3K Using Chloroalkane-Derivatized Small Molecule Inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B10.