Binding of drugs to plasma proteins, such as albumin, is a major factor which determines their pharmacokinetics and pharmacological effects. Therefore, the interactions between human serum albumin (HSA) and four antimalarial compounds selected in the 2-aryl-3H-indol-3-one series have been investigated using UV-visible, fluorescence and circular dichroism (CD) spectroscopies. Compounds produced a static quenching of the intrinsic fluorescence of HSA. The thermodynamic parameters have shown that the binding reaction is endothermic for three compounds while exothermic for the 2-phenyl-3H-indol-3-one, 3. The interaction is entropically driven with predominant hydrophobic forces with binding affinities of the order of 10(4) M(-1). The highest binding constant is observed for 3 (Kλ=280nm = 4.53 × 10(4) M(-1)) which is also the less active compound against Plasmodium falciparum. Synchronous fluorescence gave qualitative information on the conformational changes of HSA while quantitative data were obtained with CD. Displacement experiments with site markers indicated that drugs bind to HSA at site I (subdomain IIA). In addition, the apparent binding constant and the binding site number were calculated in the presence of different ions.