Rhamnolipids (RL) are surface-active glycolipids produced by Pseudomonas aeruginosa. They are always produced by this bacterium as a complex mixture of congeners, each composed of one or two rhamnose molecules linked to a dimer of 3-hydroxyfatty acids with a chain length of 8-12 carbons. Increasing interest for RL drives the need for efficient analytical methods to characterize these mixtures of molecules. High-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) is a very precise and relatively high-throughput method for the identification of each congener and their quantification in bacterial cultures. Using (13)C-labeled RL as internal standards can further enhance the precision of the quantification. Collision-induced dissociation (CID) experiments by MS/MS is a powerful tool for the detection and identification of structural variations in RL produced by various Pseudomonas strains or by a specific strain under different culture conditions. CID even allows the discrimination between isomers with subtle structural variations, like Rha-C8-C10 and Rha-C10-C8, which are almost inseparable chromatographically. We are presenting here the detailed protocols for HPLC/MS and HPLC/MS/MS analysis of RL and their lipid precursors, the 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAA), directly in bacterial culture supernatants.