Both the total amount of biothiols and thiol/disulfide ratio are wellness indicators of oxidative balance that play an important role in antioxidant defense system. Oxidized biothiols in disulfide form cannot be determined by conventional ABTS assay due to the biphasic kinetic pattern of the reaction between biothiols and ABTS radical cation (ABTS(center dot+)), necessitating the initial reduction of disulfides to thiols prior to measurement. In this study, direct simultaneous determination of biothiols (RSH) and their disulfides (RSSR) by using a single reagent of ABTS(center dot+) was achieved without preliminary chemical reduction. Thus, conventional problems of preliminary operations arising from direct borohydride reduction of disulfides to thiols, followed by formaldehyde removal of borohydride excess and complications caused by formaldehyde-thiol reactions were effectively overcome with the use of a single reagent (ABTS(center dot+)). Box-Behnken statistical experimental design was employed to specify the optimal incubation temperature and time as 60 degrees C and 60 min, respectively. The detection limits (LOD) of the proposed assay for biothiols were compared to those of the widely used DTNB (Ellman) reference assay known to be nonresponsive to disulfides, and were found to be much lower (4-70 times). The proposed biothiol assay was successfully applied to some pharmaceutical samples and synthetic serum without preliminary treatment, and the results were highly compatible with the HPLC findings. The proposed assay was demonstrated to have superior features such as simplicity, rapidity and higher sensitivity over the widely applied Ellman thiols assay.