Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of attention-deficit hyperactivity disorder (ADHD). In adults, clinical monitoring of MPH therapy is usually performed by measuring plasma MPH concentrations. In children blood sampling is however undesirable. Saliva may be an alternative matrix for monitoring MPH concentrations with the advantage that it can be obtained non-invasively. Therefore, we developed an analytical method for the quantification of MPH in both plasma and saliva. We present the validation of a liquid chromatography-tandem mass spectrometric method using a hydrophilic interaction liquid chromatography column (HILIC). In 100μL sample, proteins were precipitated with 750μL acetonitrile/methanol 84/16 (v/v) containing d9-methylphenidate as the internal standard. Standard curves were prepared over the MPH concentration range of 0.5-100.0μg/L. The total analysis time was 45s. Accuracy and within- and between-run imprecision were in the range of 98-108% and less than 7.0%, respectively. Matrix effects were greater for plasma than saliva with 46% and 8% ionization suppression. The matrix effects were adequately compensated by the use of deuterated MPH as internal standard. MPH significantly degraded in plasma and saliva at room temperature and 5°C. Samples were stable at -20°C for at least 4 weeks. The method was successfully applied for the determination of MPH concentrations in plasma and saliva samples from an adult healthy volunteer. Using protein precipitation and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry, this method allows fast, accurate and precise quantification of MPH in both plasma and saliva.