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Adalimumab (ADA) and infliximab (IFX) are therapeutic monoclonal antibodies targeting tumor necrosis factor-alpha (TNFα). They are used to treat inflammatory diseases. Clinical trials have suggested that therapeutic drug monitoring for ADA or IFX could improve treatment response and cost effectiveness. However, ADA and IFX were quantified by ELISA in all these studies, and the discrepancies between the results obtained raise questions about their reliability. We describe here the validation of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADA and IFX in human samples.Full-length antibodies labeled with stable isotopes were added to plasma samples as an internal standard. Samples were then prepared using Mass Spectrometry Immunoassay followed by trypsin digestion before ADA and IFX quantification by LC-MS/MS. ADA and IFX were quantified in serum from patients treated with ADA (n = 21) or IFX (n = 22), and the concentrations obtained were compared with those obtained with a commercial ELISA kit.The chromatography run lasted 8.6 minutes, and the quantification range was 1–26 mg/L. The method was reproducible, repeatable, and accurate. For both levels of internal quality control, for ADA and IFX, interday and intraday coefficients of variation and accuracies were all within 15%, in accordance with FDA recommendations. No significant cross-contamination effect was noted. Good agreement was found between LC-MS/MS and ELISA results, for both ADA and IFX.This LC-MS/MS method can be used for the quantification of ADA and IFX in a single analytical run and for the optimization of LC-MS/MS resource use in clinical pharmacology laboratories.