Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possi-ble biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection in-cluding simple spitting and the Salivette (R) saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was per-formed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by princi-pal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We pro-pose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.