Glutamate is the mayor excitatory neurotransmitter in vertebrate nervous system. It has a crucial role in most brain functions under physiological conditions through the activation of both ionotropic and metabotropic glutamate receptors. In addition, extracellular glutamate concentration is tightly regulated through different excitatory amino acid transporters (EAAT). Glutamate neurotransmission is also involved in the neurotoxic effects of many environmental chemicals and drugs. Furthermore, homeostatic changes in glutamate neurotransmission appear in response to prolonged block/enhancement of electrical activity. Here, we describe different approaches to evaluate alterations in glutamate neurotransmission regarding glutamate receptors and glutamate transporters by using primary cultures of neurons and astrocytes. The methods are based on the increased fluorescence of calcium-sensitive probes in response to glutamate agonists, on radioligand binding to glutamate receptors and transport sites, and on inmunocytochemistry visualization of glutamate receptors.