This work describes the development and validation of a method using High Performance Liquid Chromatography (HPLC) for Linear Alkylbenzene Sulfonate (LAS) determination in sample of wastewater from anaerobic reactors. The applied LAS standard was the dodecylbenzene sulfonic acid-sodium salt, which presents four main peaks in the chromatogram, related to different homologous of the linear alkyl chain. Different chromatographic conditions were tested with C-18, C-12 and C-8 as stationary phases, ultraviolet and fluorescence detectors, mobile phase (MP) compositions and programming time of elution gradient. The best chromatographic condition was obtained with the C-8 column, MP: methanol and of sodium perchlorate (0.075 mol L-1). The validation of this method was made with the calibration of LAS curves using water and synthetic substrate as solvents. The method was validated in order to demonstrate its precision, linearity, limit of detection of each homolog and its instrumental precision.