Background Current gold standard markers for myocardial damage are troponins I and T, which are both sensitive and specific for the detection of myocardial infarction, but require up to 6 h to become reliably elevated in serum. Investigation into markers with potential to identify patients with early ischaemic changes is therefore intense. Choline is reported to be prognostic in patients presenting with acute coronary syndromes via its release from ischaemic cell membranes. Methods Liquid chromatography tandem mass spectrometry was used to develop a method to quantitate choline in plasma and blood. The method involves addition of a deuterated internal standard to an aliquot of plasma or blood followed by organic solvent addition, which precipitates the proteins in the sample. Preparation was carried out directly into a 96-deep-well plate. Chromatography of choline used a strong cation exchange column and separation used a Waters Atlantis dC18 analytical column positioned directly before the mass spectrometer source, allowing on-line preanalytical clean up of the sample. Results The lower limit of quantitation was 0.38 μmol/L, linearity was observed up to 754 μmol/L, with a working concentration range of 0.38–224 μmol/L, inter- and intra-assay coefficients of variation were <6% and <4%, respectively. Samples were stable throughout five freeze–thaw cycles and recovery was between 94% and 114%. Conclusions The assay was successfully validated in accordance with FDA guidelines and is suitable for quantitation of choline in research and clinical settings.