Flavonoids, as well as a large number of xenobiotics, after entering circulation, bind to human serum albumin (HSA) at the IIA subdomain. This binding site is also called Sudlow site I or binding site I. Considering the large number of xenobiotics which bind to this site, it is necessary to evaluate the importance of their displacement interactions from the binding site I. Also, it is important to better describe the characteristics of flavonoid binding to the binding site I and clinical interactions with drugs which could possibly occur. In the first part of the study, characteristics of flavonoid aglycons' binding to the binding site I, as well as their most important determinants were studied. Fluorescence spectrophotometry was used to determine the binding constants and computer program Gaussian 09 was used to calculate quantum-chemical molecular descriptors. In the second part of the study, interactions between flavonoid aglycons and glycosides with selected drugs were studied by using fluorescence spectrophotometry combined with docking studies carried with AutoDock 4.2.6. computer program. Binding constants of 20 flavonoids were experimentally determined and they ranged from 5,25 × 103 to 1,95 × 105 M–1. QSAR analysis found several flavonoid features which are key determinants responsible for their binding to the binding site I: nucleophilicity and partial charge of the C3 atom, partial charge of the O4 atom, coplanarity of the A and C rings, as well as coplanarity of the AC and B rings, and HOMO and LUMO energies, with proton-donor and proton-acceptor properties and coplanarity being the most prominent ones. It was also found that the selected drugs and flavonoids can interact in binding to the binding site I, but the possibility of their mutual displacement interactions is not high as they bind to different binding regions. Flavonoid sterical and electronic characteristics which influence their binding to HSA were determined. A small possibility of displacement of selected drugs by flavonoids with clinically important consequences was found. Examined drugs can both increase and decrease fluorescence intensity of flavonoids bound to HSA, thus the application of fluorescence in routine determination of drug binding to HSA is limited.