Quantifying and characterizing Extracellular Polymeric Substances (EPS) and especially exopolysaccharides (EPSac) is an issue for understanding the hydro-physical and biological functioning of the rhizosphere. However, few comparative studies of extraction techniques have been carried out on soils and none on calcareous Mediterranean soils. Three soil-bound EPS extraction techniques, i.e. Cation Exchange Resin (CER), EDTA and NaOH + Formaldhyde (NaOH + F) were compared on three contrasted Mediterranean soils. CER presented the lowest extraction efficiency of EPSac, but also the lowest contamination of EPS by extractants and extracellular compounds. Contamination with intracellular compounds was low and similar with the three methods. Mid-Infrared (MIR) spectra enabled the best discrimination of the EPS extracts when they were prepared with CER. CER is then identified to be the suitable extraction technique of EPS (including EPSac) from soils, including calcareous soils. This technique was applied on rhizospheric and bulk soils harvested in an experimental field of tomato cultivation. A rhizospheric effect was highlighted during the growth of plants of two cultivars with both the soilbound EPSac amounts (total sugar equivalent of extracted EPS) and the MIR spectra of extracted EPS. Extraction of soil-bound EPS and their further analysis by spectral and chemometric approaches is a promising way for relating EPS chemical characteristics to their biological and hydric impacts within the plant rhizosphere in a context of agro-ecological transition and climatic change.