Exogenous and endogenous neurotoxins may have poisoning effects on living organisms. Neurotoxic signs can result from human intoxication by substances present in natural ecosystems as pollutants, such as inorganic mercury, cadmium, manganese and lead, or by abnormal accumulation of endogenous compounds, as bilirubin. Dissociated primary nerve cell cultures are powerful models that can be used to evaluate the responses of target cells at the cellular and molecular levels to the deleterious effects of neurotoxic substances. Primary cultures of nerve cells are prepared from either fetal (neurons) or 2-day-old (macroglia and microglia) rat brains, cultured with specific media. Cells can then be used to evaluate the neurotoxic effects of a particular substance. By using cells with different days-in-culture it is possible to mimic and evaluate developmental-related modifications. These modifications can comprise morphological changes, cell death by necrosis (release of lactate dehydrogenase, LDH) and apoptosis (nuclear fragmentation), altered neurotransmission (impaired uptake or increased release of glutamate), neuroinflammation (enhanced cytokine production) and the generation of oxidative damage (formation of reactive oxygen species and disruption of glutathione metabolism). Here we describe the methods for nerve cell cultures, as well as some of the procedures that can be used to assess neuronal and glial cytotoxicity induced by different neurotoxins.