The effects of amino-acid fluids on ligand binding to human serum albumin (HSA) were investigated by fluorescence and ultrafiltration techniques. Warfarin and dansylsarcosine were used as the site marker fluorescence probes for site I and site II of HSA, respectively. Amino-acid fluids specifically decreased the fluorescence intensity induced by dansylsarcosine-HSA binding without any effects on that induced by warfarin-HSA binding. The ultrafiltration technique clarified that the free fraction of the site II drug, diazepam, in human serum was increased in the presence of amino-acid fluids, while no effect was observed in the free fraction of the site I drug, warfarin. The potencies of the effect on binding to site II, observed by fluorescence and ultrafiltration techniques, correlated well with the L-tryptophan contents in amino-acid fluids or with those in L-tryptophan solutions. Based on the comparison between the effects of amino-acid fluids and L-tryptophan solutions, we confirmed that L-tryptophan in amino-acid fluids specifically inhibits drug binding to site II of HSA.