The release of reactive oxygen species (ROS) during the electron transport of mitochondrial aerobic respiration is the major source of ROS. However, contact between cells and nanoparticles (NPs) can also induce release of ROS, leading to an imbalance towards the pro-oxidative state. At low levels of ROS production, cells initiate a protective response to guarantee their survival, but an excess of ROS can damage cellular compounds such as membranes and various organelles, or directly cause genotoxicity. Thus an elevated level of ROS is an important indicator of cellular stress and an accurate recording of this parameter would be very informative. ROS can be measured by various assays, but all known assays measuring and quantifying ROS possess certain weaknesses. The problems and challenges of quantitatively detecting ROS in vitro using the 2,7-dichlorodihydrofluorescein (DCF) assay is discussed as an example. In addition, we debate the difficulties in finding a suitable and stable chemical reaction control for the DCF assay (or other ROS-detecting assays). As a conclusion, we believe that using 3-morpholinosydnonimine hydrochloride (Sin-1) as a ROS inducer in the DCF assay is feasible only qualitatively. However, a quantitative measurement of the absolute amount of ROS produced and a quantitative comparison between experiments is (at the moment) impossible.