The coformulation of the nucleos(t)ide analogs (NA) tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC) is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP). Such complexities manifest in nonlinear intracellular pharmacokinetics (PK). In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP) at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD) study among forty subjects receiving daily TDF/FTC (300 mg/200 mg) from the first-dose to pharmacological intracellular steady-state (30 days). TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC) were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM). Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV)) of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) production were 1020 fmol/106 cells (130%) and 44.4 pmol/106 cells (82.5%), resulting in (90% prediction interval) 11% (0.45%, 53%) and 14% (2.6%, 35%) reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP). Simulation-based intracellular operational multiple dosing half-lives of TFV-DP and FTC-TP were 6.7 days and 33 hours. This model described the formation of intracellular TFV-DP/FTC-TP and the interaction with dNTPs, and can be used to simulate analog:dNTP time course for various dosing strategies.