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A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for the quantitative measurement of gut microbial-derived short-chain fatty acids (SCFAs) in human infant stool has been developed and validated. Baseline chromatographic resolution was achieved for 12 SCFAs (acetic, butyric, caproic, 2,2-dimethylbutyric, 2-ethylbutyric, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic, pivalic and valeric acids) within an analysis time of 15 min. A novel sequential derivatization of endogenous and spiked SCFAs in stool via ¹²C- and ¹³C-aniline respectively, facilitated the accurate quantitation of ¹²C-aniline derivatized endogenous SCFAs based on calibration of exogenously ¹³C-derivatized SCFAs. Optimized quenching of derivatization agents prior to LCMSMS analysis further reduced to negligible levels the confounding chromatographic peak due to in-line derivatization of …