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The detection and quantitation of reactive oxygen species (ROS) receives a great deal of interest because of their importance in a wide range of physiological and pathogenic events. Probe-assisted spectroscopy (electron spin resonance, spectrophotometry, fluorescence and luminescence) is the main tool for this application. This review discusses the properties of spectroscopic probes most commonly used for ROS detection and highlights their limitations in cellular systems. These include poor stability of some probes and/or products that may be subjected to cellular metabolism and lack of specificity in their reactions with oxidants or reductants. Additional problems often arise from undesired reactions of the probes and from their non-homogeneous distribution in the studied system, production of ROS by the probes themselves, perturbation of the systems under investigation by the probes, and artifacts due to the …