The detection and quantification of hydroxyl radicals (HO•) generated by low-temperature plasmas (LTPs) are crucial for understanding their role in diverse applications of plasma radiation. In this study, the formation of HO• in the irradiated aqueous phase is investigated at various plasma parameters, by probing them indirectly using the coumarin molecule. We propose a quantification methodology for these radicals, combining spectrophotometry to study the coumarin reaction with hydroxyl radicals and fluorimetry to evaluate the formation yield of the hydroxylated product, 7-hydroxycoumarin. Additionally, we thoroughly examine and discuss the impact of pH on this quantification process. This approach enhances our comprehension of HO• formation during LTP irradiation, adding valuable insights to plasma’s biological applications.