Multicriteria optimization methodology was applied for development of isocratic reversed-phased HPLC method for simultaneous determination of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) in human urine and plasma. In the first stage of method development, pH value of the water phase, percentage of acetonitrile, temperature of the column and flow rate of the mobile phase were investigated using fractional factorial design.Afterwards, the optimal conditions were found employing central composite design and Derringer's desirability function. Two goals were considered, the retention factor of the MPAG to be in the range, between 0.8 and 1.118 which allowed well separation of MPAG from the urine and plasma peaks, and the shortest possible total analysis run time. Then, the obtained sigmoid functions were used to transform the optimization criteria into the desirability values.The satisfactory chromatographic conditions were obtained with mobile phase consisted of acetonitrile–phosphate buffer (pH 2.4; 0.04M KH 2 PO 4 ) (28:72, v/v). The separation was performed on C 18 Chromolith column (100mm×4.6mm) with flow rate of 5mL/min, the temperature of the column was 25°C and the chosen wavelength for simultaneous determination of MPA and MPAG was 215nm. The MPAG eluted at 0.552min and the duration of run was 3.092min.Afterwards, the method was subjected to validation. Linearity was observed over the concentration range of 1–50μg/mL for MPA and 1–500μg/mL for MPAG in urine and 1–60μg/mL for MPA and 1–70μg/mL for MPAG in plasma matrix. The method showed intra-day and inter-day precision with relative standard deviation lower then 5% and accuracy as recovery (%) between 100±5%.