P-glycoprotein (Pgp/ABCB1) both accounts for multidrug resistance (MDR) in chemotherapy and contributes to reduce oral bioavailability and brain distribution of drugs. Flavonoids, reported as potent Pgp inhibitors, are able to bind to the cytosolic ATP-binding site and a vicinal hydrophobic pocket. In order to explore the interaction forces governing the affinity of flavonoids towards Pgp, the 3D quantitative structure-activity relationship (QSAR) approach was used to analyze a set of flavonoid derivatives. The variation of affinity towards Pgp was investigated by VolSurf descriptors of Molecular Interaction Fields (MIFs) related to hydrophobic interaction forces, polarizability, and hydrogen-bonding capacity. The 3D linear solvation energy VolSurf model developed here identifies shape parameters and hydrophobicity as the major physicochemical parameters responsible for the affinity of flavonoid derivatives towards Pgp and hydrogen-bonding capacities as minor modulators of this activity. Furthermore, this predictive model (q(2) of 0.71) was also validated by use of an external set of 10 flavones.