An assay for the measurement of unbound L-tryptophan concentrations in plasma was developed using reverse-phase HPLC with fluorescence detection. Unbound L-tryptophan from plasma was obtained using the Amicon MPS-1 ultrafiltration device. L-tryptophan binding to the membrane in the ultrafiltration device was not significant. A linear relationship between the peak-area ratios (peak-area of L-tryptophan to internal standard) and concentrations of L-tryptophan was obtained in the range of 0.1 microgram/mL to 2.5 micrograms/mL. The intra-assay precision was less than 10% for each control (0.18 microgram/mL, 0.75 microgram/mL and 2.0 micrograms/mL). The inter-assay precision was less than 15% for each control; the accuracy for each control; the accuracy for each control, expressed as percent difference from nominal (%DFN), was less than 12%. This method was used to determine unbound L-tryptophan concentrations in plasma from nine male subjects who participated in a clinical study.