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Mycophenolic acid (MPA) is an immunosuppressive drug partly metabolized to MPA-glucuronide (MPAG), which is pharmacologically inactive. The currently available enzymemultiplied immunoassay technique (EMIT) has been reported to overestimate MPA plasma concentration in clinical samples when compared with HPLC techniques. The aims of this study were to design and validate a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique for the determination of MPA and MPAG using a low plasma volume and a simple sample preparation procedure; then to compare it with EMIT for the determination of MPA in plasma samples collected over an interdose interval at different posttransplantation periods (days 3, 7, and 30 and after 3 months) in 25 renal transplant recipients orally administered cyclosporine and mycophenolate mofetil twice daily, to investigate the origins of the differences between techniques. The LC-MS/MS technique developed showed limits of quantification (LOQs) of 0.1 mg/L and 1 mg/L for MPA and MPAG, respectively, and was linear, accurate, and precise from these LOQs up to 30 mg/L for MPA and 300 mg/L for MPAG. EMIT gave similar results to LC-MS/MS for spiked quality control samples (in a synthetic matrix or in drug-free plasma) but significantly overestimated MPA levels in clinical samples: EMIT − LC-MS/MS = +61.39% ± 57.94%, with large variations depending on patients, time elapsed since transplantation, sampling time, and concentration levels. These results confirmed the known overestimation of the EMIT assay compared with a specific method and showed that the magnitude of this overestimation depended on sampling time and time after transplantation.