L-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry (UHP–HILIC–MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7 min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600 ng/ml for carnitine and acetylcarnitine and 5–200 ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5 ng/ml for carnitine and acetylcarnitine and approximately 0.5 ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.