Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP) in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC–MS/MS method was developed, and TAF, TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 μm, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2–500 nM for TFV and TAF; 20–5000 nM for TFV-DP) with the correlation coefficients (r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE < 10.4%) and precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.