Background: The JAK/STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. Receptor-associated JAKs are activated following receptor-ligand binding. Activated JAKs phosphorylate STAT proteins, which then dimerize and translocate to the nucleus where they modulate the expression of target genes. Dysregulated JAK/STAT signaling has been implicated in the pathogenesis of multiple human malignancies. Activating mutations in JAK2 and the associated activation of STAT5 in myeloproliferative neoplasia is one example of the involvement of this pathway in human cancer. Additionally, overactivated JAK/STAT signaling has been suggested as a survival mechanism in several human cancers. Given the importance of JAK/STAT dysregulation in human diseases, it is important to identify patients with an overactivated JAK/STAT pathway for possible treatment with JAK inhibitors. Thus, we developed a gene signature assay to detect overactivated JAK/STAT5 signaling. Methods: The cancer cell line encyclopedia (CCLE) and associated gene-expression data were used to correlate the activation status of STAT5 with the induction of a set of STAT5 target genes. First, we used 27 tumor cell lines of hematologic lineage, with predetermined phosphorylated STAT5 (pSTAT5) status, to derive STAT5 activation gene signatures. Next, the putative gene signatures were validated against a different set of 13 hematologic tumor cell lines. Results: With this approach, a collection of 7 target genes were identified (PIM1, CISH, SOCS2, ID1, LCN2, EPOR, and EGR1) whose expression significantly correlated with pSTAT5 status in the 40 hematologic tumor cell lines (P < .0001), either together or in specific subsets of 4 and 6 genes (Table). Conclusions: These 4-, 6-, and 7-gene signatures can be used to stratify or select for a patient population with activated JAK/STAT5 signaling that could potentially benefit from treatments targeting the JAK/STAT5 signaling pathway.