To facilitate the evaluation of drug safety, virologic activity, and pharmacokinetics, an anion exchange isolation of tenofovir-diphosphate (TFV-DP) from human peripheral blood mononuclear cells (hPBMCs), coupled with dephosphorylation, desaltation, and detection by LC-MS-MS was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular tenofovir moieties was produced. TFV-DP was isolated from TFV-monophosphate (TFV-MP) and tenofovir (TFV), dephosphorylated with acid phosphatase to form TFV and then desalted and concentrated, making it possible for tandem mass spectral detection. An LC-MS-MS methodology was developed and validated for the determination of TFV concentrations, which directly correspond with the intra-hPBMC TFV-DP concentration. The assay was linear in the range of 50-10,000 fmol per sample. The lower limit of quantitation (LLOQ) of the method is 10 fmol per million cells with 5 million hPBMCs used. This paper outlines the development and validation of the determination of TFV-DP concentrations in femtomoles per million hPBMCs.