With the development of microbiology, Quorum sensing (QS), closely related to many physiological characteristics and activities, is getting more and more attention. QS in bacteria is a kind of mechanism for intracellular or intercellular communication in response to variation of the community in density, aiming at coordinating their population behavior and controlling gene expression. QS of Gram-negative bacteria is generally controlled by the Luxl/R-type information system, using N-acyl-homoserine lactones (AHLs) as signal molecules to regulate diverse physiological processes such as generation of exoenzyme and toxin by pathogenic bacteria, formation of biomembrane, bioluminescence, biosynthesis of antibiotic, bacteria motility and even degradation of organic pollutants. How to rapidly and accurately detect and monitor variation of AHLs in environment and cells is an important basis for the study on quorum sensing. Therefore, an overall review is presented here on AHLs as representative signal molecules, summarizing their characteristics, structures, derivatives and different analyzing methods as well. As the concentration of AHLs in the cell and the environment is usually very low, pre-concentration is necessary for its determination. For pre-treatment of liquid samples, Liquid-liquid extraction and solid-phase extraction are two common methods used to isolate AHLs from liquid matrix. The former is the most widely used one, while the latter has dual functions, concentration and purification, simultaneously. The identification of unknown signal molecules plays a vital role in understanding the information communication between microfloras. To identify structures of AHLs, the techniques of chromatograph-mass spectrometer, capillary zone electrophoresis/mass spectrometry (CZE/MS), Fourier transform ion cyclotron resonance mass spectrometry (FTICR/MS) and nuclear magnetic resonance spectroscopy (NMR) are commonly used. To quantitatively analyze AHLs compounds, the techniques of biosensors, thin layer chromatography, high performance liquid chromatography (HPLC), radioactive isotope tracer and enzyme-linked immunosorbent assay (ELISA) are used. Each technique has its own advantages and disadvantages. Biosensors are very sensitive, however the analysis of each type of AHLs needs specific strain and plasmid. Thin layer chromatography, with a lower sensitivity, is only used to detect high concentration of AHLs. Radioactive isotope tracer is immune to interference of the external environment and hence makes the pretreatment of samples simple, however, this method can only be used to determine the total, rather than specific components of AHLs. HPLC is the most widely used to measure the content and structure of AHLs, but the samples for determination need to be pretreated for purification. Being low in cost, easy to operate and high in accuracy, ELISA has turned out to be a promising method, nevertheless, it could not be used to determine molecular structure of AHLs. The determination and characterization of AHLs are very important to understand inter-microbial signal communication, however it is still a big challenge to determine AHLs qualitatively and quantitatively because they do not vary much in molecular structure and are often very low in concentration. Although the above mentioned analytic methods are quite high in sensitivity and selectivity, each has its own disadvantage. Therefore, it is advisable to combine some of these methods to bring their advantages into full play and complement each other in determining AHLs. At the same time, how to combine these methods into one that is capable of determining a variety of AHLs efficiently and rapidly shall be the focus of the researches in future. On the other hand, the current methods are mainly focused on aqueous solution. In complex natural environments, such as soil and sediment, the existence of organic and inorganic compounds simultaneously presents a great challenge to extraction, purification and determination of N-acy-homoserine lactones. It is necessary to develop some efficient methods for qualitatively and quantitatively measuring AHLs in complex matrices. The determination will play an important role in unveiling the mechanism and action of microbial quorum sensing in the natural environment, especially the soil ecosystem.