Ruthenium complexes hold a great potential in chemotherapy as an alternative to the classical platinum based drugs. The organometallic compounds studied in the present work were previously found to ex- hibit important anticancer activities. Here we have investigated the binding of three ruthenium compounds, namely [Ru(η5-C5H5)(PPh3)(bopy)][CF3SO3] 1, [Ru(η5-C5H5)(PPh3)(2-ap)][CF3SO3] 2, and [Ru(η5-C5H5)(PPh3)(isoquinpk)][CF3SO3] 3 (bopy = 2-benzoylpyridine; 2-ap = 2-acetylpyridine; isoquinpk = 1-isoquinolinyl phenyl ketone) to fatty acid human serum albumin (HSA) and fatty acid- free human serum albumin (HSAfaf) at physiological pH 7.4. The influence of the substituent groups on the heteroaromatic (N,O) coordinated ligand was also studied by fluorescence spectroscopy to get infor- mation about this binding. The Stern–Volmer quenching constants (KSV) were calculated at 293, 298 and 310 K, with the corresponding thermodynamic parameters ΔG, ΔH and ΔS as well. The fluorescence quenching method was used to determine the number of binding sites (n) and association constants (Ka) at the same temperatures. The binding site to HSA was confirmed by competitive studies of the ruthenium compounds with warfarin.