The aim of this study was to examine in vitro, by means of kinetic analysis and molecular docking simulations, the effects of the flavone diosmetin and its flavanone analog hesperetin on CYP (cytochrome P450) 2C9-mediated drug metabolism. To this purpose, the conversion of diclofenac to 4'-hydroxydiclofenac by human liver microsomes was used as a model assay for assessing the CYP2C9 inhibitory activity of these two flavonoids. Kinetic analyses showed that diosmetin and hesperetin were reversible, dead-end inhibitors of 4'-hydroxydiclofenac formation; their mean K(i) (inhibitor dissociation constant) values were 1.71 ± 0.58 and 21.50 ± 3.62 µM, respectively. Diosmetin behaved as a competitive inhibitor, since it increased markedly the K(m) (substrate concentration yielding 50% of V(max)) of the reaction without affecting the V(max) (maximum velocity of reaction). Hesperetin modified markedly K(m) and to a lesser extent also modified V(max), thus acting as a mixed competitive-noncompetitive inhibitor. The results of molecular docking simulations were consistent with those of kinetic analysis, since they showed that the putative binding sites of both diosmetin and hesperetin coincided with the CYP2C9 substrate binding site. The demonstration that diosmetin and hesperetin inhibit CYP2C9-mediated diclofenac metabolism at low micromolar concentrations is of potential clinical relevance because CYP2C9 is responsible for the biotransformation of various therapeutically important drugs that have narrow therapeutic indexes.