Objectives Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro . At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 μL/min. The analysis time was 8 min. Results For each analyte linear calibration curves were obtained over the concentration range 50-2000 μg/L, the lower limit of quantification (LLOQ) was 13 μg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 μg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.