An in-mouth headspace sorptive extraction (HSSE) procedure for the in-mouth volatile sampling of wine aroma compounds during wine tasting has been developed. The procedure is based on the application of a PDMS (polydimethylsiloxane) twister contained inside a tailored made glass tube placed into the headspace of the mouth after rinsing and spitting-off the wine, which is then followed by gas chromatography mass spectrometry analysis (GCMS). Various parameters that might affect the performance of the method (extraction time, aroma concentration) were firstly investigated. Despite the short selected in-mouth extraction time (30 s); the application of the in-mouth HSSE procedure using real wines allowed the detection of >30 volatile compounds from different chemical families in the oral cavity in a single run, and which are also present in the wine at very low concentrations. Additionally, the in-mouth HSSE profile allowed us to distinguish between wines types (with different non-volatile and volatile composition) in a similar way to that when using data from the headspace of the wine (wine-HSSE-GCMS). The simplicity, sensitivity, good repeatability and the easy automatization of this procedure, makes this technique a reliable and feasible tool to determine the chemical and biochemical changes of these compounds in the mouth in real physiological conditions providing useful -in vivo analytical data to better correlate with sensory studies.