The study investigates the interaction between human serum albumin (HSA) and three HIV reverse transcriptase inhibitors: tenofovir (TFV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF). The biophysical interactions were characterized using UV-Vis spectroscopy, steady-state and time-resolved fluorescence techniques, isothermal titration calorimetry (ITC), and in silico calculations. The results indicate that the binding between HSA and TFV, TDF, and TAF is weak, with a spontaneous ground state association. The binding is entropically driven for TFV and TDF, while TAF binding is primarily entropically dominated. The binding constant (Kd) and thermodynamic parameters obtained from ITC assays agree with those from steady-state fluorescence quenching measurements. The small internal cavity (site I) is likely the main binding pocket for TFV, while external sites (II and III) are better accommodated by TAF. Cross-docking calculations confirmed that the binding affinity of TFV and TAF can be affected by the presence of other compounds bound to albumin. The weak binding capacity of these antiretrovirals to HSA is a key factor in their low residence time in the human bloodstream, but positive cooperativity for TAF and TDF was observed in the presence of certain drugs, potentially improving their pharmacokinetic profile.The study investigates the interaction between human serum albumin (HSA) and three HIV reverse transcriptase inhibitors: tenofovir (TFV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF). The biophysical interactions were characterized using UV-Vis spectroscopy, steady-state and time-resolved fluorescence techniques, isothermal titration calorimetry (ITC), and in silico calculations. The results indicate that the binding between HSA and TFV, TDF, and TAF is weak, with a spontaneous ground state association. The binding is entropically driven for TFV and TDF, while TAF binding is primarily entropically dominated. The binding constant (Kd) and thermodynamic parameters obtained from ITC assays agree with those from steady-state fluorescence quenching measurements. The small internal cavity (site I) is likely the main binding pocket for TFV, while external sites (II and III) are better accommodated by TAF. Cross-docking calculations confirmed that the binding affinity of TFV and TAF can be affected by the presence of other compounds bound to albumin. The weak binding capacity of these antiretrovirals to HSA is a key factor in their low residence time in the human bloodstream, but positive cooperativity for TAF and TDF was observed in the presence of certain drugs, potentially improving their pharmacokinetic profile.