A sensitive and specific method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 microl was deproteinized by addition of 500 microl methanol which contained 5 microg/ml UCB 17025 as an internal standard. After centrifugation, 50 microl of supernatant was diluted with 1000 microl of 0.1% formic acid-10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 microl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C(18) 2.1 mm x 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 microg/ml to 150 microg/ml. Inter-assay inaccuracy was within +/-2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC-MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.