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Response surface method (RSM) is a set of mathematical and statistical methods for experimental design and evaluating the effects of variables and searching optimum conditions of variables to predict targeted responses. Oestrone (E1) is one of the most important steroid estrogens in sewage and waterbody. An internal standard method of HPLC-MS-MS for E1 is established with its operational parameters in the multiple reaction monitoring (MRM) mode being optimized by RSM. With a sample pretreatment using two-step solid phase extraction (SPE), the detection limit of the method was 0.5 ng/L.