Aim and applications: 1. Optimize fluorescence-based cellular assays for getting insights about mechanisms leading to cell death 2. Informative high content assays development with the aim to implement them in order to obtain preliminary toxicity information of compounds 3. Toxicity profiling of chemicals or biologicals used in screening, in particular hits behavior relative to the characterization of cell viability. Methods: Different fluorescent probes for cell viability were tested and optimized in HeLa cells by varying parameters of staining, controls and incubation times. Automated fluorescence microscopy was used to image cells in 96-well plates and automated image analysis provided tool for quantification of the response to the different assays. Image quality was estimated in each condition and ability to discriminate between positive and negative populations was statistically determined after image segmentation and features extraction. Results: Live/dead assay using calcein-AM and ethidium homodimer-1 showed significant difference in signal between living and dead cells but high variability was observed within the same population. Apoptosis probes FLICA and annexin V conjugate were tested on cells treated with staurosporine. FLICA staining gave low signal-to-background ratio and apoptotic population was not significantly discernible. Living, early apoptotic and necrotic/late apoptotic cells populations were segregated using annexin V assay. Autophagy detection using LC3B immunostaining was observable in cells treated with chloroquine. Developed image analysis method reliably segmented autophagosomes in autophagic cells but failed with dead cells. Assays were tested on a set of toxic compounds and results showed differences with information found on these substances. Conclusion: Fluorescent probes are a powerful tool to assess cell death mechanism in high content screening assay as they can report multiple biological activities at the same time. As information in this type of assay are gathered at a single cell level, high variability is expected and must be reduced as possible to successfully characterize the phenotypes encountered and reach screening requirements.