A new method for fast determination of stilbenoids from grape stems was developed. Ultrasound-assisted extraction was applied prior to chromatographic determination of stilbenoids in the extracts, and the stability of stilbenoids under extraction conditions was checked. A fractional experimental design was developed to analyze the influence on the extraction process of seven different extraction variables: temperature, ultrasound amplitude, ultrasonic cycle duration, ultrasonic probe type, time, sample-solvent ratio, and solvent (mixtures of ethanol and water). The most important variables for the recovery of major stilbenoids were studied and the final conditions optimized. With this new method, the main stilbenoids found in grape stems can be extracted in 15 min, using 75 °C as the extraction temperature and 80% ethanol as the extraction solvent, and no cleaning step with organic solvent is needed. The optimized method allowed for the analysis of stilbenoid content from 22 grape stem samples, many of them analyzed for the first time.