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Favipiravir is a broad-spectrum antiviral that is metabolised intracellularly into the active form, favipiravir ribofuranosyl-5'-triphosphate (F-RTP). Measurement of the intracellular concentration of F-RTP in mononuclear cells is a crucial step to characterising the pharmacokinetics of F-RTP and to enable more appropriate dose selection for the treatment of COVID-19 and emerging infectious diseases. The described method was validated over the range 24 – 2280 pmol/sample. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and lysed using methanol-water (70:30, v/v) before cellular components were precipitated with acetonitrile and the supernatant further cleaned by weak anion exchange solid phase extraction. The method was found to be both precise and accurate and was successfully utilised to analyse F-RTP concentrations in patient samples collected as part of the AGILE CST-6 clinical trial. • A simple, sensitive assay for quantification of T-705 RTP (F-RTP) in human PBMC. • Direct quantification of the active intracellular favipiravir metabolite (F-RTP). • Whole blood instability of F-RTP at room temperature for 1 h. • Method successfully applied to clinical samples as part of the AGILE CST-6 trial. [ABSTRACT FROM AUTHOR]