In this present study, the optimal conditions of ultrasonic-assisted extraction (UAE) of capsaicinoids from hot Chili peppers were determined for large scale preparation. First, single factor experiments were performed to optimize the extraction procedure of capsaicinoids, and initial optimized results were: ratio of solvent to mass of 6 to 10 ml/g, extraction temperature of 25 to 35°C, and extraction time of 0 to 30 min. Then, an orthogonal array experimental design (L 9 (3 4 )) was used to further optimize the extraction procedure. The results of F-test and P-value indicated that the effect order on extraction yield of capsaicinoids from high to low was ratio of solvent to mass, extraction time, and extraction temperature. The maximum extraction yield of capsaicinoids was obtained at ratio of solvent to mass of 10 ml/g, extraction time of 40 min, and extraction temperature of 25°C. Under these conditions, the extraction yields of capsaicinoids were 2.35 ± 0.042 and 3.92 ± 0.089 mg/g for conventional and UAE methods, respectively. Keywords: Chili pepper, ultrasonic-assisted extraction, capsaicinoids, orthogonal array design, single factor experiments