Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal often presents at a later stage. Gemcitabine is still an important component in PDAC treatment however there is no routine biomarker to predict its efficacy. We had previously performed a discovery cohort of two separate RNA-blood signatures in 60 patients (NCT00789633) showing an association with PFS and OS. Called GemciTest, this CE-IVD molecular test requires 2.5ml whole blood sample before starting the patient's 1st line chemotherapy. From this liquid biopsy, IVD measures the expression levels of nine genes using real-time PCR processes. This abstract presents the clinical validation of GemciTest. Methods: In this study, clinical validations were done on 214 patients (mean 68.7-year-old; 37-88) from 3 distinct cohorts with the University of Wisconsin Biobank, GemciPANC trial (NCT03599154), and BACAP (NCT02818829). These cohorts included first-line treatment,with either a gemcitabine or fluoropyrimidine based regimen. Results: Patients with a clinical benefit response identified by GemciTest (31.5%) had a significantly longer progression free survival (PFS) (5.4 months vs. 3.1 months p = 0.0032) and a longer overall survival (OS) (13.1 months vs. 5.4, p < 0.0003). In multivariate analyses including tumor localization and performance status, this signature continued to be associated with PFS (HR = 0.52 (0.34–0.81) p = 0.003) and OS (HR = 0.44 (0.28 – 0.69) p = 0.0002).Conclusions: GemciTest validation was performed, showing a strong association with PFS and OS.