Rationale Glyphosate is one of the most widely used herbicides and it is suspected to affect the intestinal microbiota through inhibition of aromatic amino acid synthesis via the shikimate pathway. In vitro microbiome bioreactors are increasingly used as model systems to investigate effects on intestinal microbiota and consequently methods for the quantitation of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in microbiome model systems are required. Methods An optimized protocol enables the analysis of both glyphosate and AMPA by simple extraction with methanol:acetonitrile:water (2:3:1) without further enrichment steps. Glyphosate and AMPA are separated by liquid chromatography on an amide column and identified and quantified with a targeted tandem mass spectrometry method using a QTRAP 5500 system (AB Sciex). Results Our method has a limit of detection (LOD) in extracted water samples of <2 ng/mL for both glyphosate and AMPA. In complex intestinal medium, the LOD is 2 and 5 ng/mL for glyphosate and AMPA, respectively. These LODs allow for measurement at exposure‐relevant concentrations. Glyphosate levels in a bioreactor model of porcine colon were determined and consequently it was verified whether AMPA was produced by porcine gut microbiota. Conclusions The method presented here allows quantitation of glyphosate and AMPA in complex bioreactor fluids and thus enables studies of the impact of glyphosate and its metabolism on intestinal microbiota. In addition, the extraction protocol is compatible with an untargeted metabolomics analysis, thus allowing one to look for other perturbations caused by glyphosate in the same sample.