Novel analytical methods were designed for monitoring humic acid photodegradation in environmental waters. Modified CUPric Reducing Antioxidant Capacity (CUPRAC) spectrophotometric and chromatographic methods were used for the determination of dihydroxybenzoate isomers (DHBAs) produced from a salicylate probe, which was hydroxylated by hydroxyl radicals ((OH)-O-center dot) produced from the photodegradation of humic acid under ultraviolet A-radiation. The combined use of CUPRAC colorimetry and HPLC was shown to effectively monitor humic acid photodegradation and (OH)-O-center dot generation for the first time. The formation of 2,5-dihydroxybenzoate and 2,3-dihydroxybenzoate, as major and minor products, respectively, from the hydroxylation of a salicylate probe was demonstrated by HPLC and confirmed by a modified CUPRAC method to indicate (OH)-O-center dot formation from humic acid, which acted as both a generator and absorber of hydroxyl radicals. Salicylate hydroxylation showed an increase between 30 and 50 min of illumination, and was affected by the initial concentration of humic acid up to 0.01% but not by solution pH around the neutral values. Traces of Fe(III) and Mn(II) present in natural waters decreased the (OH)-O-center dot production, but EDTA partly restored the probe hydroxylation by chelating these metal cations. Since humic acid-mediated (OH)-O-center dot generation may aid in natural disinfection processes, this work may extend our comprehension of concentration- and time-dependent generation of (OH)-O-center dot in environmental waters and of the possible effects of other antioxidants.