Serum albumin (SA) acts as a reservoir and transport protein for endogenous (e.g. long chain fatty acids (LCFAs) and chloride anion (Cl–)) and exogenous ligands (e.g. drugs) in the blood. In this study, the alteration of the nonspecific binding constant (K values) of chlorothiazide (CTZ) to bovine serum albumin (BSA) induced by the presence of oleic acid (OLA) and/or Cl– was evaluated by a second derivative spectrophotometric method at simulated physiological conditions (pH = 7.4 and 37 ⁰C). The residual background impacts caused by the incomplete suppression of BSA signals have been entirely eliminated in the second derivative spectra. Since the therapeutic effects of a drug is closely related to its free fraction in the blood, which in turn may be altered in the presence of endogenous ligands, the results of the present study are very useful for providing safer and efficient pharmacological information in clinical settings.