Previous work from our group has shown that the single thiol in human serum albumin can be oxidized with hydrogen peroxide and peroxynitrite to a remarkably stable sulfenic acid (HSA-SOH). Up to the moment there are few techniques available to measure sulfenic acid. We have developed a suitable technique based on the reaction of sulfenic acid with the chromophoric thiol thionitrobenzoate (TNB). The latter was synthesized from DTNB by reduction with excess 2-mercaptoethanol followed by ion exchange chromatography. Indeed, TNB reacted with HSA-SOH formed from the reaction of HSA-SH (0.5 mM) with hydrogen peroxide (4 mM, 4 min, 37°C). The decrease in TNB absorbance at 412 nm was biphasic, suggesting the presence of two consecutive reactions. The first one was assigned to the reaction of HSA-SOH with TNB to form a mixed disulfide and the second, to the reaction of the mixed disulfide with TNB to form HSA-SH and DTNB. From the fit of the kinetic run to a double exponential function, it was possible to determine the rate constants of both reactions as 104 ± 9 M^-1 s^-1 and 5 ± 1 M^-1 s^-1 for the first and second reactions, respectively, at pH 7.4 and 25ºC. The amplitude of the first phase increased linearly with HSA-SOH concentration and allowed us to quantify the amount of HSA-SOH formed per mole of HSA as 0.12 ± 0.01, representing 0.30 ± 0.02 HSA-SOH per oxidized thiol. When HSA was exposed to a higher concentration of hydrogen peroxide (15 mM, 4 min, 37°C) the amount of HSA-SOH per mole of HSA decreased from 0.12 to 0.05, suggesting that the thiol was overoxidized to sulfinic or cisteic acid. Neither native HSA-SH nor HSA blocked with N-ethylmaleimide reacted with TNB. To further characterize the reaction, we determined the rate constant of the reaction of DTNB with HSA-SH, the reverse of the second reaction, as 14.8 ± 0.8 M^-1 s^-1. In order to have and independent estimate for the forward second reaction, we tried to prepare HSA-STNB by incubating HSA-SH with DTNB, but our attempts were unsuccessful as we obtained more than one molecule of TNB per albumin molecule. We are currently using the reaction with TNB to study the stability, reactivity and properties of HSA-SOH.