Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor growth and aggressiveness. Endoplasmic Reticulum (ER) stress has been documented in most major cancers, and the ability to tolerate persistent ER stress through an effective unfolded protein response enhances cancer cell survival, angiogenesis, metastasis, drug resistance and immunosuppression. The ER stress sensor IRE1α contributes to tumor progression through XBP1 mRNA splicing and regulated IRE1α-dependent decay of mRNA and miRNA. The aim of this study was to perform a molecular characterization of series of tumor samples to explore the impact of intratumoral IRE1 signaling in non-small cell lung cancer characteristics. To monitor IRE1 splicing activity, we adopted a fragment length analysis to detect changes in the length of the XBP1 mRNA before and after splicing as a method for measuring sXBP1 mRNA levels in tumors because sXBP1 mRNA is not probed by standard transcriptomic analyses. We demonstrate for the first time that XBP1 splicing is a valuable marker of lung cancer aggressiveness, and our results support a model in which IRE1 downstream signaling could act as a regulator of Epithelial to Mesenchymal Transition (EMT). Our findings study highlights the role of IRE1α downstream signaling in non-small cell lung cancer and opens a conceptual framework to determine how IRE1α endoribonuclease activity shapes the EMT program.