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A quantitative, non-destructive cellular autofluorescence based in vitro imaging assay has been developed and applied to study the cytotoxicity of Sodium Lauryl Sulfate (SLS) and HgCl 2 on Balb/c 3T3 cells. A phenomenological double logistic model was proposed to quantify and relate the observed kinetic changes of fluorescence to the toxic potency of chemical compounds. This work forms the basis for cellular autofluorescence measurements in in vitro toxicity screening assays.