Cultivated rice (Oryza sativa) is a global staple food that subsists calories for more than half of the world’s population. Emerging evidence supports that a number of bioactive compounds have been identified in rice including tryptophan and its derivatives (oxitriptan, serotonin, tryptamine and auxin). The development of single optimized extraction of bioactive compounds from plant may complicate due to the structural diversity. A new ultrasound-assisted extraction (UAE) of tryptophan and its derivatives has been optimized and validated.Experiment with a three-level and six-factor Box-Behnken Design (BBD) combined with Response Surface Methodology (RSM) was employed to evaluate the significances of studied factors (solvent composition,temperature, solvent-solid ratio, pH, amplitude and cycle) on the extraction yields. Subsequently, extraction kinetic was studied to confirm satisfactory recovery of the extracted compounds from the matrix. Later, by chromatographic analyses, tryptophan, oxitriptan, serotonin, tryptamine and 3-indole acetic acid (auxin) were identified and quantified in the extracts of rice grains. A complete validation of the method was performed and presented agreeable linearity (correlation coefficients higher than 0.9998) with high extraction precisions (in average, expressed as CV, repeatability and intermediate precision were lower than 2.0% and 2.6% respectively). The method was successfully applied in the extraction of a number of real samples. Hence, it can be concluded that UAE under optimum conditions can be considered as a powerful tool for the extraction of tryptophan and its derivatives from a wide variety of rice grains.