A medicinal chemistry approach combining in silico and in vitro methodologies was performed aiming at identifying and characterizing putative allosteric drug-binding sites (aDBS) at the interface of the transmembrane- and nucleotide-binding domains (TMD-NBD) of P-glycoprotein. Two aDBSs were identified, one in each NBD, by means of in silico fragment-based molecular dynamics, and characterized in terms of size, polarity, and lining residues. From a small library of thioxanthone and flavanone derivatives, experimentally described to bind at the TMD-NBD interfaces, several compounds were identified to be able to decrease the verapamil-stimulated ATPase activity. An IC50 of 81 ± 6.6 μM is reported for a flavanone derivative in the ATPase assays, providing evidence for an allosteric efflux modulation in P-glycoprotein. Molecular docking and Molecular Dynamics gave additional insights on the binding mode on how flavanone derivatives may act as allosteric inhibitors.