Background: Immune checkpoint inhibitors (ICIs) are associated with durable antitumor activity in advanced non-small cell lung cancer (NSCLC), however, selection of the best candidates for this type of therapy is a challenge. PD-L1 expression, the only adequately validated predictive biomarker of response to ICIs, is imperfect, and not completely discriminatory between responders and non-responders. Gene expression profile (GEP) is a promising way to evolve in this field. The objective of this study was to identify a GEP of immune-oncology related genes, predictive of response to ICIs in NSCLC. Methods: This study analyzed two cohorts of advanced NSCLC patients treated with ICIs in any line: a discovery cohort composed of 66 Brazilian patients and a validation cohort composed of 54 Spanish patients. Clinical data were collected from medical records. Total RNA was extracted from FFPE tumor tissue. Gene expression profile (GEP) was assessed using the nCounter PanCancer IO360 panel (NanoString Technologies), which comprises 770 genes involved in the cancer immune response. LASSO regression with Cox proportional hazards approach was used to define the new gene expression signature. Results: In the discovery cohort, median age was 62.5 years (36 - 81 years). Most patients were males (57.6%), stage IV (91.9%), ECOG-PS≤1 (90.7%), smokers/former smokers (83.3%) and had adenocarcinoma histology (67.7%). The majority was treated with ICI in the first-line setting (42.4%) and nivolumab was the most frequently used drug (52.6%). At data cutoff, median follow-up for overall survival (OS - time from ICI initiation to death by any cause) was 27.7 months and for real-world progression-free survival (rwPFS - time from ICI initiation to disease progression as assessed by treating physician) was 28.2 months. The median OS and rwPFS were 17.7 months and 5.6 months, respectively. We identified a 6-gene signature (Lung Tumor Score – LungTS) that discriminate patients at low and high risk for death (median OS: 36.1 vs 15.5 months; p < 0.001) and for disease progression (median rwPFS: 10.1 vs 4.2 months; p < 0.001). Considering the expression of CD274 (PD-L1), the LungTS signature also discriminated two populations regarding OS and rwPFS (p < 0.0001). In the validation cohort, which had clinicopathological characteristics similar to the discovery cohort, the OS for high versus low LungTS was 17.0 months and 9.0 months, respectively (p = 0.004). Patients with high score also showed better rwPFS, although not statistically significant (median 10.0 versus 4.0 months; p = 0.11). Conclusions: Our study identified a novel 6-gene immune-signature predictive of response to ICI in NSCLC.