OBJECTIVE To develop a rapid assay for determining tryptophan in human serum. METHODS The serum sample was injected into the high performance liquid chromatography (HPLC) system after the precipitation of protein with perchloric acid, and was monitored with a fluorometric detector with wavelength settings of 254 nm for excitation and 313 nm for emission. The mobile phase is 20% acetonitrile. RESULTS Good linearity was observed in 1.22-400 mumol.L-1 range for tryptophan. The detection limit was 0.1 mumol.L-1. The intra- and inter-assay coefficients of variation (CV) were 2.57% and 3.66%, respectively. The average recovery was 97.13%. The detection time of serum tryptophan was within 5 min. Phenyalanine, tyrosine, serotonin, and 5-hydroxyindoleacetic acid didn't interfere in the assay. CONCLUSIONS This method is simple, rapid, sensitive, accurate, and suitable for clinical application.