Specimens obtained with endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) are often tiny and fragmented leading to an inconclusive and doubtful diagnosis. To overcome the limitations of EUS-FNA in the cytological diagnosis of pancreatic adenocarcinoma (PCA), we evaluated whether quantification of the S100P protein combined with EUS-FNA reliably discriminated between PCA and benign pancreatic lesions (BPL). A high sensitivity sandwich ELISA for S100P protein was developed to aid in the detection of PCA in small samples obtained using EUS-FNA. After experimental verification of the sandwich ELISA with cell lines and mouse xenograft tumors, 27 consecutive patients with suspicious PCA who underwent EUS-FNA were enrolled in the present study examining the combination of S100P protein assessment and EUS-FNA cytology. The concentration of the S100P protein in EUS-FNA samples from the PCA group was significantly higher than that in the BPL group (P=0.04). Using receiver operating characteristic curve analysis, we determined the S100P protein cut-off value for PCA diagnosis to be 99.8 ng/ml. The S100P protein levels combined with EUS-FNA cytology to detect PCA showed the following diagnostic values: sensitivity, 94.4% [95% confidence interval (CI), 75.7-99.1%]; specificity, 88.9% (95% CI, 51.8-99.7%); positive predictive value, 94.4% (95% CI, 72.7-99.9%); negative predictive value, 88.9% (95% CI, 51.8-99.7%); accuracy, 92.6% (95% CI, 75.7‑99.1%); and area under the curve, 0.92 (95% CI, 0.79-1.00). We established a novel quantitative analysis for the S100P protein in EUS-FNA samples which, when combined with EUS-FNA cytology, could provide promising results for the reliable diagnosis of PCA.