Oxidative stress is associated with the pathogenesis of many diseases. To better understand this, it is often necessary to measure relative levels of oxidative stress in cells. Biochemical methods can be highly quantitative but tend to be undynamic given they usually require fixation or destruction of the cell. Oxidative stress-sensitive fluorophores such a CellROX, can be used in living tissue and with certain apparatus which permit dynamic measurement. To do this in single cells currently requires the use of flow cytometry; which isn’t suitable for many cell types, or conventional photometry; which is laborious and limits time course of single experiments. To overcome these limitations, we developed the use of the Biotek Cytation cell imaging system with CellROX fluorophores. Here, we present the early optimisation of the technique. A HEP G2 cell line was maintained in culture and plated in 96 well plates. Cells were loaded with 5µM CellROX deep red for 30 minutes. A Cytation cell imaging system (Biotek, USA) was used to capture brightfield and fluorescent images. To model oxidative stress, we exposed cells to 20 µM hydrogen peroxide for 30 min. To measure the effectiveness of our optimisation steps, relative fluorescence was quantified using ImageJ software. Fluorescent values are given as mean ± S.E. We first tested the effect of CellROX loading order. When cells were loaded before exposure to H2O2­, we observed no increase in fluorescence. However, we observed a considerable increase in fluorescence when cells were loaded after exposure to H2O2­ (Control (n=162); 1.15 ± 0.07, Before (n=104); 1.00 ± 0.07, After (n=104); 2.05 ± 0.18, MAU, p < 0.001. Our next experiment sought to establish the effect of well cell density on fluorescent intensity. Exposure to H2O2 produced an inverse sigmoidal relationship between cell density (250 – 30,000 cells per well) and fluorescent intensity (n = 104, p < 0.001). For subsequent experiments we used a density of 5000 cells per well, which offered an ideal practical compromise. Our final experiment was designed to test the sensitivity of the method. Cells were exposed to a range (0-50 µM) of H2O2 concentrations which produced a concentration-dependent increase of fluorescence, which saturated at 20µM (n = 90, p < 0.001). These data suggest the Cytation cell imaging system when used with CellROX fluorophores may offer a convenient way to measure oxidative stress in living cells. CellROX loading order and cell density are important considerations for experimental design. Our next experiments will optimise the technique for use with primary cardiac myocytes.