The interactions between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) were investigated by measuring fluorescence anisotropy, poly-dispersity index, and time-resolved fluorescence. Also, isothermal titration calorimetry (ITC) was performed. The fluorescence spectra of the drugs exhibited an appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. The gradual addition of HSA led to a marked increase in fluorescence anisotropy (r), and fromthis value it is argued that the drugs were located in a restricted environment of the protein. The binding constants for the ASA–HSA and CYC–HSA complexes were found to be 1.27 9 108 and 4.23 9 108 molL-1, respectively, as calculated from the relevant fluorescence data. The polydispersity index and size distribution of the protein–drug complex weremeasured at several concentrations of the drugs by the zeta potential technique,which confirmed the already obtained experimental results. From the analysis of the steady-state and time-resolved fluorescence quenching of the drugs in aqueous solutions in the presence of HSA, it was found that the quenching is static in nature. ITCexperiments revealed that, in the absence of drugs, the dominant forces are electrostatic,whereas hydrophobic andweak electrostatic forces became significant in the presence of the drug.The primary binding pattern between the drugs andHSAwas interpreted as a combined effect of hydrophobic association and electrostatic interactions.

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