In this work, an automatic headspace in-tube extraction (ITEX) method for the accurate determination of highly polar volatile compounds from wine has been developed and validated. Only 20 microliters of wine diluted 1:20 and containing three internal standards are put in a 2mL vial, heated at 60°C and extracted with 100 0.5mL pumping strokes. The validation of the method showed satisfactory figures of merit. The extraction efficiency calculated by multiple extractions from the same sample ranged from 85–99%. The method can constitute a reliable alternative for the analysis of these compounds in many other matrices.