Visualization of NADH by fluorescence microscopy makes it possible to distinguish mitochondria inside living cells allowing structure analysis of these organelles in a noninvasive way. Mitochondrial morphology is determined by the occurrence of mitochondrial fission and fusion. During normal cell function mitochondria appear as elongated tubular structures. However cellular malfunction induce mitochondria to fragment into punctiform, vesicular structures. This change in morphology has also been associated with the generation of reactive oxygen species (ROS) and early apoptosis. In this study we demonstrated that autofluorescence imaging of mitochondria in living eukaryotic cells provides structural and morphological information that can be used to describe mitochondrial health. We firstly established the illumination conditions that do not affect mitochondrial structure and calculated the maximum safe light dose that the cells can be exposed to. Subsequently we showed that by sequential recording of mitochondrial fluorescence it is possible to monitor changes in mitochondrial morphology in a continuous non-destructive way. This approach was then applied to describe mitochondrial toxicity induced by potential toxicants exposed to mammalian cells. Both mouse and human cells were used to evaluate mitochondrial toxicity of different compounds with different toxicities. It has been proven in this study that this technique constitutes a novel approach to explore chemical induced toxicity due to its reliability to monitor mitochondrial morphology changes and corresponding toxicity in a non-invasive way.