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An LC–MS/MS method for measurement of TAF and TFV in human plasma and CSF was developed.The method is fully validated to simultaneously measure TAF and TFV.Lower limits of quantification of 0.5 ng/mL in plasma and 0.1 ng/mL in cerebrospinal fluid.The method was applied to patient plasma and cerebrospinal fluid samples.A liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of tenofovir and tenofovir alafenamide concentrations in human plasma and cerebrospinal fluid. Tenofovir and tenofovir alafenamide were extracted from matrix by solid phase extraction. The dried extraction eluents were dissolved in water for LC–MS/MS analysis. Separation was achieved with a Phenomenex Synergi 4 μm Polar-RP 80A column (50 × 2 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 5 min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 0.5 to 500 ng/mL for the plasma assay and 0.1–50 ng/mL for the cerebrospinal fluid assay. The intra- and inter-day accuracy and precision were less than 12% in low, medium, and high quality control samples for both matrices. The validated methods were applied to the analysis of plasma and cerebrospinal fluid samples of a patient undergoing tenofovir therapy which involved the switch from Stribild® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg) to Genvoya® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir alafenamide 10 mg).