A method was developed for determination of total tryptophan content in soy- and milk-based nutritional products. The method uses enzymatic (pronase) digestion of the protein to release tryptophan, which is separated and quantitated by isocratic reversed-phase liquid chromatography with fluorescence detection. Enzymatic digestion is completed for products containing these types of proteins in less than 6 h and is accomplished under chemically mild conditions (pH 8.5,50°C), which do not significantly degrade tryptophan. Chromatographic separation is complete in about 8 min, including an internal standard. The precision of the method is 1-2% relative standard deviation. Accuracy is demonstrated by agreement with theoretical values for standard proteins (amino acid sequence known) and by quantitative recoveries of overspikes, which use either free tryptophan or a standard protein as the spiking material. The method allows determinations on samples containing a wide range of tryptophan values. Appropriate sample size selection and verification of digestion time requirements should allow the method to be applied to different protein types as well. The method allows 24 h turnaround of tryptophan analyses, and quantitative recoveries represent a significant improvement on existing techniques applied to infant formulas and other nutritional products.