Serotonin is an important neurotransmitter produced mainly in intestines, and the serotonin level in feces can be an indicator for intestinal environment. Human feces, however, contain a large amount of contaminants, which vary widely owing to food contents and intestinal environment, and these contaminants interfere with the determination of serotonin levels in human feces. To remove these contaminants and determine serotonin levels, we developed a new method using solid phase extraction (SPE) and column-switching LC-MS/MS. Stable isotope-labeled serotonin was added to human feces samples prior to SPE as an internal standard to correct individual differences of the matrix effect. The recovery rate of SPE was 55.9–81.0% (intraday) and 56.5–78.1% (interday) for feces from two subjects. We analyzed 220 fecal samples from 96 subjects including 76 pregnant and post-delivery women. The endogenous serotonin content per unit weight of dried feces was 0.09–14.13 ng/mg for pregnant and post-delivery women and 0.30–9.93 ng/mg for the remaining.