Tryptophan is a nutritionally essential amino acid for both humans and animals. Besides acting as a building block for protein synthesis, tryptophan (Trp) and its metabolites are crucial for maintaining neurological function, immunity, and homeostasis in the body. To uncover the regulatory role of Trp and its metabolites in cell nutrition, metabolism and physiology, various analytical methods, including high-performance liquid chromatography (HPLC), have been developed to determine key Trp metabolites. Here we describe a rapid and sensitive method for the simultaneous analysis of Trp and its metabolites along with other amino acids by HPLC involving in-line pre-column derivatization with o-phthaldialdehyde (OPA) and dual-channel fluorescence detection. OPA reacts very rapidly (within 1 min) with Trp, 5-hydroxytryptophan, 5-hydroxytryptamine, and tryptamine at room temperature (e.g., 20-25 °C) in an autosampler. Their derivatives are immediately injected into the HPLC column without the need for extraction. Trp metabolites that cannot react with OPA but are fluorescent can be detected by setting the excitation and emission wavelengths of the fluorescence detector in another detection channel. The autosampler is programmed to mix Trp and its metabolites with OPA for 1 min to generate highly fluorescent derivatives for HPLC separation and detection (Channel A, excitation = 270 nm and emission = 350 nm; Channel B, excitation = 340 nm and emission = 450 nm). The detection limit for Trp and its metabolites is 30 pmol/mL or 150 fmol/injection. The total time for chromatographic separation (including column regeneration) is 55 min for each sample. Our HPLC method can be used for the analysis of amino acids (including Trp) in alkaline protein hydrolysates and of Trp and its metabolites in biological samples.