The concentration of reserpine in urine was directly and quantitatively measured by using the excitation-emission fluorescence (EEM) combined with threeway calibration methodologies.Two calibration methods are based on the alteating trilinear decomposition (ATLD) and the self-weighted alteating trilinear decomposition (SWATLD) algorithms,respectively.These chemometric methodologies have the second-order advantage,which is the ability to get accurate concentration estimates of interested analyte(s) even in the presence of uncalibrated interferences.The satisfactory results on spiked urine samples are obtained,when the component number was chosen to 3 (N=3) for both the methods.This experiment is easily carried out without time-consuming and complicated pretreatment.It has proved that the three-way calibration methodologies based on ATLD and SWATLD can be feasible to directly quantify the medical content of reserpine in urine.