Efforts to understand microbiome-host interactions in disease development and progression are growing, with short-chain fatty acids (SCFAs) produced by gut bacteria recognized as key metabolic mediators. To facilitate studies on the effect of bacterially secreted metabolites on human cells, we have developed a new LC-MS(/MS) method that allows quantitative analysis of underivatised SCFAs and simultaneous non-targeted screening for additional microbial or endogenous metabolites from cell culture media. Comprehensive method optimization resulted in methanolic gradient elution using a Kinetex XB-C18 column allowing for quantitative QQQ-MS analysis of propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate, using deuterated internal standards. The method was validated in terms of linearity, selectivity, stability as well as inter- and intra-day accuracy and precision. To integrate non-targeted metabolomics, the assay was subsequently transferred to an LC-QTOF-MS platform and cross-validated to ensure accuracy and precision of SCFA analysis. Applying this method to study the effects of the breast cancer drug tamoxifen and 10 human tamoxifen metabolites (desmethyltamoxifen, Z-endoxifen, (E/Z)-4′-hydroxy-desmethyltamoxifen, (E/Z)-4-hydroxytamoxifen, tamoxifen-N-glucuronide, (E/Z)-tamoxifen-4-glucuronid, (E/Z)-DM-tamoxifen-4-O-glucuronide, (E/Z)-4-hydroxytamoxifen-N-glucuronide, (E/Z)-endoxifen-4-sulfate, (E/Z)-tamoxifen-4-sulfate) on the secretome of faecal bacterial communities revealed a pronounced impact of the parent drug and non-sulfated metabolites. The omission of derivatisation leads to fast and simple sample preparation. Together with the short LC run time (10 min) and the comprehensive information obtained by combining targeted and non-targeted metabolomics in a single run, the new method represents a valuable tool for the in vitro investigation of metabolite-mediated microbiome-host interactions.