Pharmaceutical interactions with human serum albumin (HSA) are of great interest, because FSA is a pharmacokinetic determinant and a good model for exploring the protein ligand interactions, Due to their hydrophobic nature, naturally occurring flavones, which possess various pharmacological activities, bind to HSA in human plasma. Here, we have identified the binding modes of two representative flavones baicalin (BLI) and its aglycon, baicalein (BLE) to HSA using a combination of experimental and computational approaches. The association properties were measured by applying spectroscopic methods, and a higher affinity was found for BLE. As evidenced by displacement and chemical unfolding assays, both ligands hind at Sudlow site I. Furthermore, molecular docking was utilized to characterize the models of HSA flavone complexes, and molecular dynamics (MD) simulations as well as free energy calculations were undertaken to examine the energy contributions and the roles of various amino acid residues of HSA in flavones binding; the mechanism whereby glycosylation affects the association was also discussed. The present work provides reasonable binding models for both flavones to HSA.