The differential diagnosis of biliary stenosis is a critical problem for gastroenterologists. An early identification of malignant lesions would enable the rapid resort to surgical resection which currently represents the only potentially curative option. Unfortunately, the diagnostic value of all available methods (e.g. imaging technics, standard serum biomarkers) is limited by relatively poor accuracy and negative predictive value. Recently, our group and others highlighted new potential cancer biomarkers in bile by using comparative proteomic analysis. Nevertheless, to date, only a few candidates have been verified for their diagnostic performances in discriminating between malignant and non-malignant stenoses. In addition, no data have yet been collected on the simultaneous measurement of these proteins with the intent of evaluating the diagnostic interest of a panel of biomarkers. To overcome the limitation of classical verification tools and give a new impetus to the translation of bile biomarkers into clinical diagnostics, mass spectrometry-based quantification could represent a rapid and cost-effective opportunity thanks to its capacity for multiplexed, high-throughput analysis, combined with its analytical specificity and reliable quantification. Here we developed the first Selected Reaction Monitoring (SRM) assay for the multiplexed measurement of cancer biomarkers in human bile. For this purpose, 8 potential biomarker candidates previously highlighted by proteomic analysis were selected. Equal volumes of bile collected from patients presenting with malignant and non-malignant biliary stenosis were stacked on the top of a SDS-PAGE gel. Proteins were then digested in-gel with trypsin and proteotypic peptides of each candidate biomarker were quantified by nanoLC-SRM on a 5500-QTrap mass spectrometer (ABSciex) using heavy synthetic peptides as standards (PEPotecTM, Thermofisher). SRM data were finally analysed using Skyline software and manual validation. The developed assay proved to be valuable and reliable to quantify all the selected candidates. Moreover, the results confirmed the simultaneous overexpression of some of the proteins in bile samples from malignant stenoses. Overall, our data demonstrate the ability of SRM to quantify cancer biomarkers in human bile and emphasize the interest of using multiplexed SRM assays to assess the diagnostic potential of a panel of bile biomarkers in differentiating biliary stenoses. Work supported by the PRIME-XS consortium.