The bacterial composition of the gut has been found to affect many diseases, including several gastrointestinal cancers. The microbiome appears central in the production of certain metabolites that enter circulation, especially those from bile acids and the essential amino acid tryptophan. The tumor-microenvironment may also produce changes in metabolites, such as those from the tryptophan-kynurenine pathway, of which several compounds may be measured in the blood. As data emerges from large scale metabolomics studies, there will be a need to validate metabolomic biomarkers to confirm their clinical utility. This task also requires knowledge about biological variation of the same metabolites in a healthy population. For this purpose, a novel method was developed for quantification of bile acids and tryptophan metabolites in samples of human serum by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Salting-out assisted liquid-liquid extraction was optimized with the ion-pairing reagent trifluoroacetic acid. In this way, both polar tryptophan metabolites and non-polar bile acids could be extracted with a high recovery, favorable matrix effects, and improved chromatographic focusing, by using straightforward robot pipetting. The instrumental analysis was fast (4 min and 32 s) and with sample injections done directly from the extraction microplate. The method was applied to quantify metabolites in serum from healthy probands, and for investigating inter- and intraindividual variations over six hours.