BACKGROUND For therapeutic drug monitoring (TDM) of mycophenolic acid (MPA), which is frequently proposed, saliva might be a suitable and easy-to-obtain biological matrix. The study aimed to validate an HPLC method with fluorescence detection for determining mycophenolic acid in saliva (sMPA) in children with nephrotic syndrome. METHODS The mobile phase was composed of methanol and tetrabutylammonium bromide with disodium hydrogen phosphate (pH 8.5) at a 48:52 ratio. To prepare the saliva samples, 100 µL of saliva, 50 µL of calibration standards, and 50 µL of levofloxacin (used as an internal standard) were mixed and evaporated to dryness at 45 °C for 2 h. The resulting dry extract was reconstituted in the mobile phase and injected into the HPLC system after centrifugation. Saliva samples from study participants were collected using Salivette® devices. RESULTS The method was linear within the range of 5-2000 ng/mL, was selective with no carry-over effect and met the acceptance criteria for within-run and between-run accuracy and precision. Saliva samples can be stored for up to 2 h at room temperature, for up to 4 h at 4 °C, and for up to 6 months at - 80 °C. MPA was stable in saliva after three freeze-thaw cycles, in dry extract for 20 h at 4 °C, and for 4 h in the autosampler at room temperature. MPA recovery from Salivette® cotton swabs was within the range of 94-105%. The sMPA concentrations in the two children with nephrotic syndrome who were treated with mycophenolate mofetil were within 5-112 ng/mL. CONCLUSIONS The sMPA determination method is specific, selective, and meets the validation requirements for analytic methods. It may be used in children with nephrotic syndrome; however further studies are required to investigate focusing on sMPA and the correlation between sMPA and total MPA and its possible contribution to MPA TDM is required.