AbstractEBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV‐associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK‐2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV‐associated carcinomas. A sensitive and reliable method was developed to quantify VK‐2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK‐2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK‐2019, two purported metabolites, and the internal standard, VK‐2019–d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK‐2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5–500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK‐2019 was stable in plasma at −70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK‐2019 in a patient who received a single and multiple oral daily doses of 120 mg.