Distal cholangiocarcinoma is an aggressive malignancy with a dismal prognosis. There is a lack of diagnostic and prognostic biomarkers. An improved understanding of proteomic changes associated with malignancy and identification of potential biomarkers can help improve the survival of patients. A workflow utilizing discovery mass spectrometry and verification by parallel-reaction monitoring was used to analyze surgically resected formalin-fixed paraffin embedded samples from distal cholangiocarcinoma patients and normal bile ducts in order to identify differentially expressed proteins. 20 tumors and 6 controls were successfully analyzed in the discovery experiment and 16 tumors and 9 controls in the PRM analysis. In the discovery experiment a total of 3057 proteins were identified. 87 proteins were found to be differentially expressed between the conditions (q<0.05 and fold change ≥2 or ≤0.5). 31 proteins were upregulated in the distal cholangiocarcinoma samples as compared to controls and 56 downregulated. Bioinformatic analysis revealed an abundance of the differentially expressed proteins to be associated with the tumor reactive stroma. Parallel-reaction monitoring verified 28 proteins as upregulated and 18 as downregulated. In conclusion several proteins without prior association with cholangiocarcinoma biology were identified and verified as differentially expressed between distal cholangiocarcinoma samples and normal bile ducts. These proteins can be further evaluated to elucidate their biomarker potential and role in distal cholangiocarcinoma carcinogenesis.