An analytical ultrasound-assisted extraction (UAE) technique has been optimized and validated for the extraction of tryptophan and its derivatives from rice grains. A Box–Behnken design in conjunction with a response surface methodology based on six factors and three levels was used to evaluate the effects of the studied factors prior to optimizing the UAE conditions. The significant (p < 0.05) response surface models with high coefficients of determination were fitted to the experimental data. The most significant (p < 0.0001) effect is the solvent-to-sample ratio while quadratic effects caused by temperature and solvent-to-sample ratio were of moderate importance (p < 0.05). The optimal UAE conditions were as follows: extraction time of 5 min, ultrasound amplitude of 30%, cycle of 0.7 s−1, extraction temperature of 30 °C, 8% methanol in water as the extraction solvent at pH 3 and a solvent/solid ratio 5:1. The method validation ensured that appropriate values were obtained for the LOD, LOQ, precision and recovery. Furthermore, the method was successfully applied to the analysis of a number of rice samples of different varieties. It was demonstrated that this particular UAE method is an interesting tool for the determination of tryptophan and tryptophan derivatives in rice grain samples.