Quantification of hydroxyl radical concentration using two chemical probes was assessed through the Fenton reaction. The probes were 1,2-benzopyrone (coumarin) for fluorescence and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for electron spin resonance (ESR). The corresponding hydroxylated species, namely 7-hydroxycoumarin (7HC) and 2-hydroxy-5,5-dimethyl-1-pyrroline-N-oxide (DMPO-OH adduct), were monitored by fluorescence and ESR-spin trapping techniques, respectively. The experiments were designed according to the theoretical conditions determined for stable fluorescence and EPR signals. The results demonstrate that: the optimal [chemical probe] : [H₂O₂] ratio predicted by a simplified quasi-steady-state model was in good agreement with the optimal [chemical probe] : [H₂O₂] ratio observed experimentally for [H₂O₂] : [Fe²⁺] = 10, and the proper adjustment of the [chemical probe] : [H₂O₂] ratio at a given concentration of the Fenton's reagent improves the detected amount of hydroxyl radicals. Finally, using DMPO required a higher concentration compared to coumarin to yield the same amount of ˙OH detected but resulted in a more reliable probe for detecting ˙OH under the consideration of this study.