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Rational dosing of antibiotics in neonates should be based on pharmacokinetic (PK) parameters assessed in specific populations. PK studies of neonates are hampered by the limited total plasma volume, which restricts the sample volume and sampling frequency. Available drug assay methods require large sample volumes and are labor-intensive or time-consuming. The objective of this study was to develop a rapid ultra-performance liquid chromatographic method with tandem mass spectrometry detection for simultaneous quantification of amoxicillin, meropenem, cefazolin, cefotaxime, deacetylcefotaxime, ceftriaxone, and vancomycin in 50 microl of plasma. Cleanup consisted of protein precipitation with cold acetonitrile (1:4) and solvent evaporation before reversed-phase chromatographic separation and detection using electrospray ionization tandem mass spectrometry. Standard curves were prepared over a large dynamic range with adequate limits of quantitation. Intra- and interrun accuracy and precision were within 100% +/- 15% and 15%, respectively, with acceptable matrix effects. Coefficients of variation for matrix effects and recovery were <10% over six batches of plasma. Stability in plasma and aqueous stocks was generally sufficient, but stability of meropenem and ceftriaxone in extracts could limit autosampler capacity. The instrument run time was approximately 3.50 min per sample. Method applicability was demonstrated with plasma samples from an extracorporeal membrane oxygenation-treated neonate. Different beta-lactam antibiotics can be added to this method with additional ion transitions. Using ultra-performance liquid chromatography mass spectrometry, this method allows simple and reliable quantification of multiple antibiotics in 50 microl of plasma for PK studies of neonates.