Introduction Truffles have an important economic value due to their gastronomic qualities appreciated in “grande cuisine”. While Perigord and White Alba truffles are well-valued, Burgundy truffle (Tuber uncinatum) is not well-characterized in its production area. INRA is involved to help the producers to better characterize this truffle through different research axes, especially the influence of the ripeness and the geographical origin on aromatic composition. For this purpose, we had first to define an analytical strategy to better characterize aroma compounds in this noble fungus. Methods Burgundy truffles were analyzed by Solid Phase MicroExtraction (SPME) and Dynamic HeadSpace (DHS) coupled with Gas Chromatography - Mass Spectrometry (GC-MS) for volatile organic compounds (VOC) identification. A new in vitro analytical method by Proton Transfer Reaction – Mass Spectrometry (PTR-MS) (4) was developed to obtain an aroma mass fingerprint of all the samples. In addition, sensory analyses were performed by a trained panel. Results Truffles are very aromatic, and 1 g of fungus is sufficient for GC-MS analyses. The DHS parameters were defined through a kinetic study: an incubation temperature of 36°C for 15 min was selected. The influence of the trapping and drying steps were studied to set the best parameters and to enable the identification of more than 70 compounds. The SPME technique allowed to better extract the most volatile compounds but does not allow to identify as many compounds. Depletion of hydronium ions was observed by PTR-MS during the analysis of 25 mg of truffles. While it is not possible to weigh less without generating an error in the repeatability, a headspace dilution system with a humidified airflow has been developed to permit VOC analysis. After integrating the peak areas, the data were compiled to observe the evolution of the areas depending on time and incubation temperature. An analysis of variance (ANOVA) was performed to determine the significant differences between the different conditions and to propose the best methodology for fingerprint analysis. Conclusions This study enable the development of an analytical methodology to better characterize the Burgundy truffle. It is part of a broader project aimed at (i) Correlate the data from PTR-MS, GC-MS and sensory analyses to define what is a quality truffle (ii) Cross this results with genetic and microbiota investigations for a scientific contribution to the constitution of an IGP (Protected Geographical Indication) request. Novel Aspect This work leaded to methodological development for in vitro analysis of truffle by PTR-MS. It also allowed to create a new analytical protocol for DHS on our Gerstel equipment.