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Methane-producing Archaea can be found in a variety of habitats, including the gastrointestinal tract, where they are linked to various diseases. The majority of current monitoring methods can be slow and laborious. To facilitate gut methanogenic Archaea detection, we investigated flow cytometry for rapid quantification based on the autofluorescent F420 cofactor, an essential coenzyme in methanogenesis. The methanogenic population was distinguishable from the SYBR green (SG) and SYBR green/propidium iodide (SGPI) stained background microbiome based on elevated 452 nm emission in Methanobrevibacter smithii spiked controls. As a proof-of-concept, elevated F420-autofluorescence was used to detect and quantify methanogens in 10 faecal samples and 241 in vitro incubated faecal samples. The methanogenic population in faeces, determined through Archaea-specific 16S rRNA gene amplicon sequencing, consisted of Methanobrevibacter and Methanomassiliicoccus. F420-based methanogen quantification in SG and SGPI-stained faecal samples showed an accuracy of 90 and 100% against Archaea proportions determined with universal primers. When compared to methane and Archaea presence, methanogen categorisation in in vitro incubated faeces exhibited an accuracy of 71 and 75%, with a precision of 42 and 70%, respectively. To conclude, flow cytometry is a reproducible and fast method for the detection and quantification of gut methanogenic Archaea. [ABSTRACT FROM AUTHOR]