A method for the determination of microcystins concentrations (MC-LR, MC-RR, MC-YR) in natural water samples was optimized using High Performance Liquid Chromatography with UV/PDA detection after Solid Phase Extraction. Solid Phase Extraction is needed to clean natural sample and concentrate pollutant. The method was validated by evaluation of specificity and repeatability. Average recoveries in ultra-pure grade water were better than 95% with Relative Standard Deviation values lower than 4%. Matrix interferences, as pH, conductivity and organic matter content, were tested. pH must be fixed between 6 and 8 to avoid under-estimation or over-estimation and conductivity did not interfere with the analytical method. Organic Matter content negatively impacted microcystins quantification unlike organic matter characteristics. It over-estimated the concenration by an average of 19%. Then, the developed method was applied to study the occurrence of microcystins in Pigeard pond (France). These results constitute the first report on the concentration levels and seasonal variations of microcystins in this resource water.