In this study, a novel spectrofluorometric assay based on p-cresol (4-methyl phenol) probe is developed for the measurement of HOBr scavenging activity. It is the first study involving the use of a p-cresol probe for the determination of the HOBr scavenging activity of biothiols. While the p-cresol probe (lambda(ex) = 260 nm, lambda(em) = 305 nm) has fluorescence characteristics, its brominated derivatives emerging at the end of the oxidation reaction with HOBr do not show fluorescence. The initial fluorescence intensity of the p-cresol probe is decreased in the presence of the brominating agent, HOBr, and this decrease is lower in the presence of HOBr scavenging antioxidants. The scavenging activities of biothiols tested with respect to the developed method decrease in the following order: penicillamine > N-acetyl cysteine > L-glutathione (reduced) > cysteamine > homocysteine > glutathione ethyl ester > cysteine > 1,4-dithiothreitol > lipoic acid > methionine. Penicillamine (IC50 = 10.12 mu M) was the most effective HOBr-scavenger among the tested biothiols. The results obtained with the developed method for biothiols and some pharmaceutical samples were statistically compared (using ANOVA) to those found by the reference methods (KI/taurine and UPLC). The advantage of the proposed method over the KI/taurine assay was demonstrated.