The development of a rapid, reproducible and simple method of extraction of the majority capsaicinoids (nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin) present in hot peppers by the employment of ultrasound-assisted extraction is reported. The study has covered four possible solvents for the extraction (acetonitrile, methanol, ethanol and water), the optimum temperature for extraction (10–60°C), the extraction time (2–25min), the quantity of sample (0.2–2g), and the volume of solvent (15–50mL). Under the optimum conditions of the method developed, methanol is employed as solvent, at a temperature of 50°C and an extraction time of 10min. The repeatability and reproducibility of the method (R.S.D.<3%) have been determined. The capsaicinoids extracted have been analysed by HPLC with fluorescence detection and using monolithic columns for the chromatographic separation. The method developed has been employed for the quantification of the various capsaicinoids present in different varieties of hot peppers cultivated in Spain.