We developed a method for the simultaneous detection and quantification of tryptophan and the eleven tryptophan metabolites indole, indole-3-methyl, indole-3-carboxaldehyde, indole-3-acetic acid, indole-3-propionic acid, indole-3-acrylic acid, kynurenine, kynurenic acid, tryptamine, serotonin, and 3,3’-diindolylmethane in fecal water extracts. Fecal water extracts were prepared by diluting feces one in ten (w/w) with Dulbecco’s Phosphate Buffered Saline. After centrifugation the supernatants were filtered through a 0.45 µm filter and diluted one to one with a 10% (w/v) zinc sulfate solution to precipitate proteins. After centrifugation 20 µL of sample was injected into a liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) instrument. Separation was achieved by using a C18 column and a mobile phase consisting of water, methanol and formic acid (0.1%), with a gradient ranging from 5% to 95% methanol in 45 minutes. Mass spectra were acquired in positive full scan mode in the mass range of 50 to 1700 m/z, and by cycling through different fragmentation energies to acquire information on molecular and fragment ions. Quantification was performed by using a calibration curve of standards. Tryptophan and its metabolites were detected with limits of detection (LODs) and limits of quantification (LOQs) ranging from the low nM range up to 50 nM. In theory, this approach can also be used to assess tryptophan and its metabolites in serum and other biological matrices.