The invention belongs to the technical field of endogenous substance detection and discloses a GC-MS (gas chromatography-mass spectrometer)-based method for quantifying eleven types of short-chain fatty acids in intestinal contents and fecal samples. By adoption of a hydrochloric acid solution prepared from saturated salt water for acidification, high-stability ethyl acetate for extraction and N-tert-butyldimethylsilyl-N-methyl trifluoroacetamide MTBSTFA for derivatization, the eleven short-chain fatty acids in mouse intestinal contents and fecal samples are quantified by a gas chromatography-mass spectrometer. The method is high in accuracy and precision and low in detection limit, can be used for quantitative detection of eleven types of short-chain fatty acids and can meet detection requirements on determination of major short-chain fatty acids in the mouse intestinal contents and the fecal samples. Moreover, the method is conducive to analysis and comparison of metabolic disposition laws of the short-chain fatty acids under normal and enteritis conditions and observation whether changes of the short-chain fatty acids can represent dynamic conditions of enteritis or not, thereby providing significant bases for potential biomarkers under the enteritis condition.