Abstract The poly (ADP-ribose) polymerase, tankyrase 1, is an encouraging pharmacological target for cancer therapy. First discovered by its association with the TRF1, tankyrase 1 plays an important role in the maintenance of telomere length, sister telomere association, and mitotic spindle organization. More recently, interactions between tankyrase 1 and axin were shown to stabilize β-catenin and modulate Wnt signaling, enabling selective targeting of the Wnt pathway with tankyrase inhibitors. In addition, tankyrase 1 has also demonstrated potential as a target for synthetic lethality for BRCA-deficient cancers. Given the effect on these cancer-associated pathways, there is a growing interest for identifying tankyrase-specific inhibitors. Historical methods to evaluate tankyrase activity are not amenable for high throughput screening. Limited availability of the full length enzyme required radioactivity to attain the needed sensitivity. Truncation of tankyrase 1 improved solubility and increased availability, but the physiological response may not be equivalent to the full length enzyme. Many of these assays have also used modified substrates where molecules such as fluorescein or biotin are linked to NAD without characterizing the effects on enzyme activity. Most historical assays have also relied on measuring autoribosylation of tankyrase 1; whereas, transribosylation may be more relevant for assessing the activity of tankyrase 1 on other molecules. To address these issues, we have developed a novel, in vitro, high throughput assay for evaluating the transribosylation activity of tankyrase 1. This is accomplished using an ELISA format which semi-quantitatively detects poly (ADP-ribose) (PAR) deposited onto immobilized histone proteins by tankyrase 1. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate, and HRP substrate generate a colorimetric or chemiluminescent signal, and the conversion of substrate correlates with tankyrase 1 activity. This assay ensures physiological significance by utilizing the full length enzyme and unmodified substrate in a transribosylation format, providing sensitivity down to pmols of enzyme. This assay is utilized to demonstrate the specificity of the tankyrase 1 inhibitor, XAV939, for tankyrase 1 compared to PARP1 and to reveal differential effects of activated DNA for full-length tankyrase 1 compared to the truncated enzyme. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3884. doi:1538-7445.AM2012-3884