This study aimed to determine the ability of single fiber reflectance (SFR) spectroscopy incorporated in endoscopic ultrasound fine needle biopsy (EUS-FNB) procedures in the pancreas to distinguish benign and malignant pancreatic tissue in patient with pancreatic masses suspected for malignancy. Methods: This study was designed as a prospective observational single center study and included consecutive adult patients, who were scheduled for EUS-FNB of a solid pancreatic mass suspected for pancreatic ductal adenocarcinoma (PDAC). In total, seven optical parameters, derived from the absorption acquired spectra, were analyzed: blood volume fraction (BVF), microvascular saturation, average vessel diameter, bilirubin concentration (BIL), Mie amplitude, Mie slope and Rayleigh amplitude. Results: Forty-five patients with a suspicious pancreatic lesion undergoing EUS-FNB were included, of which most of the patients (N=34) were ultimately diagnosed with PDAC. Finally, 27 out of 45 (60.0%) patients were used for the final analysis of the optical parameters. The median (IQR) BVF differed significantly in benign compared to malignant tissue (0.86 [0.30-2.03] and 4.49 [1.28-15.47]; p=0.046). Combining BVF and BIL to a new parameter (θ) improved the discrimination between PDAC and benign pancreatic tissue (p=0.026). The area under the curve of θ was 0.84, resulting in a 92.8%, 75.0%, 97.5%, 50.0% and 91.3% sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for detection of PDAC. Conclusion: Differentiation between PDAC and benign pancreatic tissue using SFR spectroscopy during EUS-FNB procedures is promising. Future work should focus on comparing the diagnostic performance combining SFR spectroscopy with EUS-FNB and EUS-FNB alone.