A label-free colorimetric method for the detection of biological thiols (biothiols) was developed.This method is based on prevention of the photoinduced reduction of auric ions (Au(III)) in thepresence of amino acids (acting as a reducing agent) by biothiols; the photoinduced reduction isinhibited due to the strong interaction of the biothiols with Au(III). In this method, the samplewas first incubated in an assay solution containing Au(III) and threonine; the sample solutionwas then exposed to 254 nm UV light. For samples without biothiols, this process led to thephotoreduction of Au(III) followed by growth of gold nanoparticles accompanied by the visuallydetectable development of a red coloration typified by an absorption peak at ca 530 nm.Conversely, in the presence of biothiols, reduction of Au(III) to Au(0) was prevented byentrapment of Au(III) within the biothiols via the thiol group. The solution thus remainedcolorless even after UV irradiation, which was used as an indicator of the presence of biothiols.Using this strategy, biothiols were very conveniently analyzed by monitoring color changes ofthe samples with the naked eye or a UV–vis spectrometer. The strategy based on this interestingphenomenon exhibited high selectivity toward biothiols over common amino acids and wassuccessfully employed for reliable quantification of biothiols present in human plasma,demonstrating its great potential for clinical applications.