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We evaluated whether pancreatic main duct fluid can provide protein biomarkers with prognostic value.Mass spectrometry proteomics was applied to as little as 20µL of fluid collected at the time of tumor surgical resection. Biomarker proteins identified for 27 patients were correlated with clinical outcomes.Thirteen patients had pancreatic ductal adenocarcinomas, 4 had intraductal papillary mucinous neoplasm with in situ adenocarcinoma, 5 had ampullary adenocarcinomas, 2 had intraductal papillary mucinous neoplasms, and 3 had benign diseases. In pathologic stage II or higher pancreatic ductal adenocarcinoma, moderate or high expression of S100A8 or S100A9 proteins was associated with a median disease recurrence–free survival of 5.8 months compared with 17.3 months in patients with low expression (P = 0.002). Median overall survival was 12.6 versus 27 months for patients with moderate to high versus low S100A8 and A9 expression (P = 0.02).This analysis suggests distinct proteomic signatures for pancreatic cancer. Patients in our study with elevated levels of S100A8 or A9 in the ductal fluid, a near absence of pancreatic enzymes, and high levels of mucins were found to have significantly worse prognosis. Although further validation is needed to corroborate these findings, analysis of pancreatic ductal fluid is a promising tool for identifying biomarkers of interest.