The quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called auto-inducers. Those molecules regulate bacterial gene expression, including resistance or virulence ones. Our study focuses on the auto-inducing N-acyl-homoserine lactones (AHLs) of Gram-negative bacteria from the Burkholderia and Paraburkholderia genus. The AHLs dier in their acylchain length and may be modied by a 3-oxo or 3-hydroxy substituent, or various degrees of unsaturation (1). As the bacterial signal specicity depends on all of these features, including the unsaturation position, it is mandatory to determine their complete structural characterization to better understand the population regulation and virulence phenomenon. Therefore, this study aimed to develop a method enabling the localization of the C=C double bond while using signicantly low amount of biological material. Our method is based on LC-MS/MS or/and SFC-MS/MS analyses of bacterial extracts after in-solution derivatization by a Paterno-Buchi reaction which is a photochemical (2+2) addition of a ketone on a C=C double bond leading to the formation of an oxetane ring (2). To optimize the in-solution derivatization of AHLs, the influence of various parameters (UV wavelength, solvent, Paterno-Buchi reagent, AHL/reagent ratio, reaction time) was studied with a design of experiments on 12.5 pmol of an AHL standard per LC-MS/MS analysis. The best results were obtained after a 15-minute derivatization at 254 nm, in acetonitrile, and with 2-acetylpyridine at AHL/reagent ratio of 1/100. MS/MS of Paterno-Buchi products enabled the localization of the C=C double bond due to the specic fragmentation of the oxetane ring. As a proof of concept, LC-MS/MS and SFC-MS/MS analyses were performed on bacterial extracts after in-solution derivatization. Finally, the implementation of the Paterno-Buchi reaction post-column is currently under investigation. (1) Churchill et al., Chem. Rev., 2011, 111 (1), 68-85 (2) Xu et al., Anal. Chim. Acta, 2018, 1028, 32-44