Scanning laser microscopy (SLM) was used to develop an assay to visualize the generation of intracellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U-87,663 on ROS formation. Cultured N18 neuroglioma cells were challenged by extracellular addition of cumene hydroperoxide, and subsequent intracellular generation of ROS was characterized by measuring the fluorescence intensity of the ROS indicator 2',7'-dichlorofluorescein (DCF). The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U-87,663, can be most accurately assessed if results from individual cell clusters are analyzed independently. It is possible and necessary to account for the these experimental and analytical properties in order to characterize the properties of the antioxidant activity precisely. We determined that the temporal increase in DCF fluorescence was consistent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U-87,663. The rate constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U-87,663. These differences in reaction rates combined with differences in the cellular distribution of the ROS indicator DCF, the antioxidant U-87,663, and the bulk of the ROS prevented detection of any protection of U-87,663 may offer.